CA2643700A1 - High throughput genome sequencing on dna arrays - Google Patents

Abstract

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new. e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both "upstream" and "downstream" of the adaptors, identification of entire target sequences may be accomplished.

Description

HIGH THROUGHPUT GENOME SEQUENCING ON DNA ARRAYS
CROSS-REFERENCE TO RELATED APPLICATIONS

[00011 This application claims priority to provisional applications Ser. No.
60/776,415, filed February 24, 2006, which is hereby incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

100021 This application has been partially funded by the Federal Government through Grant No. I U01 AI057315-01 of the National Institute of Health.

BACKGROUND OF THE INVENTION

100031 L,arge-scale sequence analysis ofgenomic DNA is central to understanding a wide range of biological phenomena related to states of health and disease both in humans and in many economically important plants and animals, e.g. Collins et al (2003), Nature, 422: 835-847; Service, Science, 311: 1544-1546 (2006); Hirschhorn et al (2005), Nature Reviews Genetics, 6: 95-108; National Cancer Institute, Report of Working Group on Biomedical Teclutology, "Recommend'ation for a Human Cancer Genome Project," (February, 2005);
Tringe et al (2005), Nature Reviews Genetics, 6: 805-814. The need for low-cost high-throughput sequencing and re-sequencing has led to the development of several new approaches that employ parallel analysis of many target DNA fragments simultaneously, e.g.
Margulies et al, Nature, 437: 376-380 (2005); Shendure et al (2005), Science, 309: 1728-1732; Metzker (2005), Genome Research, 15: 1767-1776; Shendure et al (2004), Nature Reviews Genetics, 5: 335-344; Lapidus et al, U.S. patent publication US
2006/0024711;
Drmanac et al, U.S. patent publication US 2005/0191656; Brenner et al, Nature Biotechnology, 18: 630-634 (2000); and the like. Such approaches reflect a variety of solutions for increasing target polynucleotide density in planar arrays and for obtaining increasing amounts of sequence infotmation within each cycle of a particular sequence detection chemistry. Most of these new approaches are restricted to determining a few tens of nucleotides before signals become significantly degraded, thereby placing a limit on overall sequencing efficiency.

SUBSTITUTE SHEET (RULE 26) 100041 Another limitation of traditional high-throughput seqaencing techniques is that 30 random positioning of DNA targets over an arrav surface, which is used in many sequencing methods, reduccs the packing effciencv of those targets from what is possible by attaching.
DNA at predefined sites such as in a grid.
100051 In view of such limitations, it would be advantageous for the field if an additional approach were available to increase the amount of sequencing information that couid be 35 obtained from an array of target poivnucleotides. Another need in the art is for an efficient and inexpensive kvay to prepare array supports with billions of binding sites at sLibmicron sizes and distances.

SUMMARY OF THE INVENTION

[00061 Accordingly, in one aspect, the invention addresses the problems associated with 40 short sequence read-lengths produced by many approaches to large-scale DNA
sequencing, including the problem of obtaining limited sequence information per enzymatic cycle. Also provided are methods and compositions for preparing random arrays of engineered nucleic acid niolecules able to support billions of molecules, including molecules at submicron sizes and distances.

45 [0007] In one aspect, the invention provides a method of determining the identification of a first nueleotide at a detection position of a target sequence, wherein the target sequence comprises a pluraiity of detection positions. In a preferred aspect, the method includes two steps: providing a plurality of concatemers and identifying the first nucleotide. Each concatemer comprises a plurality of monomers, and each monomer comprises: {i) a first 50 target domain of the target sequence comprising a first set of target detection positions; (ii) a first adaptor comprising a Type lis endonuclease restriction site; (iii) a second target domain of the target sequence comprising a second set of target detection positions;
and (iv) a second interspersed adaptor comprising a T~,rpe Iis endonuclease restriction.
site. In a preferred embodiment, the target sequence concatemers are immobilized on a surface. In a 55 further er.abodirnent, the surface is functionalized.
[(10081 In one embodiment, the invention provides amethod of determining the identification of a first nucleotide at a detection position of a target sequezzce in which the identify~ing step comprises contacting the concatemers with a set of sequencing probes. In an exemplary embodiment, the sequencing probes each comprise a first domain 60 corraplemcntarv to one of tl;G a~l ,ptc,~s, a unique nucleotide at a first interroaatinn positicana as ~.<a preferr tbe C

sequencing probes is accomplished under conditions such that if the unique nncleotide is complementary to the first nucleotide, a sequencing probe b.vbridizes to the concatemer, thereby identitying the first nucleotide.
6 5 [00091 Ix another embodiment, each adaptor comprises an anchor probe, a hybridization site and an identifyirrg, step. The identifying step in an exemplary embodiment comprises:
hybridizing anchor probes to anchor probe hybridization sites, hybridizing sequencing probes to tar-et detection positions adjacent to the adaptors, ligating adjacent hybridized sequencinty and anchor probes to forzn ligated probes, and detecting the ligated probes to 7 0 identifNr the first nucleotide.
[0010] In another embodiment, each adaptor comprises an anchor probe hybridization site, and the identifying step comprises hybridizing anchor probes to the anchor probe hvbridization sites and adding a polymerase and at least one dNTP comprising a label. `l'he polymerase and the at least on dNTP are added under conditions whereby if the dNTP is 75 perfectly complementary to a detection position, the dNTP is added to the anchor probe to form an extended probe, thereby creating an interrogation position of the extended probe.
The first nucleotide is identif ied by determining the nucleotide at the interrogation position of the extended probe.
1001.11 In a further embodiment of the invention, a nucleotide at a second detection so position is identified. In still further embodiments o1:'the invention, nucleotides at a third detection position, at a fourth detection position, at a fi:fih detection position, and/or at a sixth detection position is identified.
[0012[ In one embodiment, the invention provides a method of'determining the identification of a first nucleotide at a detection position of a target sequence, wherein the 85 target sequence the target sequence concatemers are immobilized on a surface, and that surface comprises functional moieties including but not limited to amines, silanes, and hydroxyls. In a further embodiment, the surface comprises a plurality of spatially distinct regions comprising said irnmobilized concatemers. In a still further embodiment, the concatemers are immobilized on the surface using capture probes.
90 [O01.3[ In one aspect, the invention provides a substrate comprising, a pluralitv of irnmobilized concatemers, each monomer of said concatemer comprising: a first target sequence, a first adaptor comprising a Type Ils endonuclease restriction site, a second target sequence, and a second interspersed adaptor comprising a Type IIs endoniclease restriction s tc. TI-!,- Type IIs -;;str`tction sitc,-If''~c flj_-~st adaptoi` rra-v or may bi, the P~ 1 _vond .,_ In embodiment, each monomer further comprises a third target sequence and a third interspersed adaptor comprising a Type IIs endonuclease restriction site, and in a still further embodiment, each monomer further comprises a fourt.h target sequence and a fourth interspersed adaptor comprising a Type IIs endonuclease restriction site.
o [0014] In anotber aspect, the invention provides methods for inserting multiple adaptors in a target sequence. In a preferred aspect, the method includes the steps of:
(i) figatizzg, a first adaptor to one terminus of said target sequence, wherein the adaptor comprises a binding site for a restriction enzvme, circularizing the product from step (i) to create a first circular palynucleotide; cleaving the circular polynucleotide with a restriction enzyme, wherein the 105 restriction enzyme is able to bind to the binding site within the first adaptor; ligating a second adaptor, wherein said second adaptor comprises a binding site for a restriction enzyme; and circularizing the product from step (iv) to create a second circular polynucleotide. In some embodiments, steps (iii) through (v) are repeated to insert a desired number of adaptors in the target sequence. In a preferred embodiment, the circularization 110 step comprises adding a Cirel~igaseTM enzyme.
100151 In another embodiment, the circularization step eoniprises adding a circularization sequence to a second terminus of the target sequence, hybridizing a bridge template to at least a portion of the adaptor and a portion of the circularization sequence, and ligating the first and second termini together to circularize the target sequenee.
115 [0016] In another aspect, the invention provides a method for identifying a nucleotide sequence of a target sequence. In this method, a plurality of interspersed adaptors is provided within the target sequence, and each interspersed adaptors has at least one boundary with the tar(,ret sequence. At least one nucleotide adjacent to at least one boundary of at least two interspersed adaptors is identified, thereby idetitifying the nucleotide 120 sequence oftbe target sequence.
100171 In yet another aspect, the invention provides a library of polynucleotides. [n a preferred aspect, the library comprises more than one nucleic acid fragment, and each fragment comprises a plurality of interspersed adaptors in a predetertnined order. Each interspersed adaptor has at least one end that comprises a sequence which is not able to 125 cross-hybridize with other sequences of` other interspersed adaptors of the plurality. In a further prei:erred aspect, the predetermined order of interspersed adaptors is identical for every nucleic acid fra-ment.

[0I3I.81 In ort : ~, ' p)vides a r--`hod for identl' .,_ '. ,,.
s ~, .. S

130 from each of a plurality of fragments of the target polynucleotide and forming a random array of the amplicons, hybridizing one or more sequencing probes to the random array.
determining the identity of at least one nucleotide adjacent to at least one interspersed adaptor by extending the one or more sequencing probes in a sequence specific reaction, and repeating the b.ybridization and identifying steps until a nucleotide sequence of the target 135 polynueleotide is identiried. In a preferred aspect, the sequencing probes are hybridized to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on interspersed adaptors. In a preferred aspect, each fragment contains a plurality of interspersed adaptors at predetermined sites. In a further aspect, each amplicon comprises multiple copies of a 140 fragment in numbers such that the fragments substantially cover the target polynucleotide.
In a still further aspect, the amplicons of the random array are fixed to a surface at a density such that at least a majority of the amplicons is optically resolvable.
[0019] In another aspect, the invention provides a method of identifying a nucleotide sequence of a target sequence which comprises the steps of providing a random array of 145 concatemers, hybridizing one or more probes from a first set of probes to the random array, hybridizing one or more probes from a second set of probes to the random array, ligating probes forni the f-irst and second sets which are hybridized to a target concatemer at contiguous sites, identifying the sequences of the ligated first and second probes, and repeating the hybridizing, ligating and identifyin.g steps until the sequence of the target 150 sequence is identified. In a preferred aspect, the random array of concatemers comprises concatemers fixed to a planar surface having an array of optically resolvable discrete spaced apart regions, and each concatemer comprises multiple copies of a fragment of the target polynucleotide, such that the number of different concatemers is such that their respective fragments substantially cover the target sequence. In a further aspect, each discrete spaced 155 apart region has an area of less than l~em', such that substantially all the discrete spaced apart regions have at most one concatemer attached.
100201 In still another aspect, the invention provides a method of identifying a nucleotide sequence of a target sequence which comprises generating a plurality of coneatemers cornprising multiple copies of a fragrnent of the target sequence, forming a random array of 160 the concatemers fixed to a surface at a density such that at least a majority of the concatemers are optically resolvable, and identifying a sequence of at least a portion of each fr t7.-me#Tt adi'rwe,.nt to ~"* l. a,~4 int.--;rersed adaptor at least one c<3ncate.rner, thereby .~~gei s BRIEF DESCRIPTION OF THE DRAWINGS

165 1002I1 Figs. IA- iG illitstrate the invention aDd applications thereof.
[00221 Pigs. 2A-2G illustrate various methods of insertin~.= adaptors in a nucleic acid fragment to produce a target polynacleotide containing interspersed adaptors.
[0023] Figs. 3A-3E illustrate a method of high-throughput sequencing that can be implemented on target polyrZucleotides containing interspersed adaptors.
170 100241 Fig. 4 provides a comparison of structured and standard random DNA
arrays made by attaching RCR products.
[0025] Fig. 5 illustrates reference pat#erns on. an ordered array, t00261 Fig. 6 shows random arrays imaged on a rSBH instrument.
100271 Fig. 7 shows three array images overlaid with slight shifts for easier viewing.
175 [0028] Fig. 8 shows five array images overlaid with slight shifts.
100291 Fig. 9 shows five array images overlaid with slight shifts.
[00301 Fig. 10 shows an image of an array in which lines of capture probe across the surface of the coverslip were used to specifically bind to DNBs.

DETAILED DESCRIPTION OF THE INVENTION

180 100311 The practice of the present invention may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and immunology, which are within the skill of the art. Such conventional techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using a label.
185 Specific illustrations of suitable techniques can be had by reference to the example herein below. Ilowever. other equivalent conventional procedures can, of course, also be used.
Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysas: A Laboratory _;Vlanual Series (Vols. I-IV), Using Antibodies: A
Laboratory Manual, Cells: A Laboratory Nfcznucrl, PCR PrameY: A Lahoratory 11arrucal, and 190 .1-folecular Cloning: A Laboratc3ry, :Llarruul (all from Cold Spring Flarbor Laboratory Press).
Strver, L. (1995) I3ioeherrais/ry (4th Ed.) Freeman. New York. Gait, :-C)lt`gorauclcotide Synthesis: A Prac tical APPraac h" 1984, IRL Press, L.ondQn, Nelson and Cox (2000'), Lehninger, Principles of Biocliemistry-.* 3a E;d., W. H. Freeman Pub., New York, N.Y. and Berg et al. {2002)Biochemistry, 5th Ed., W. H. Freeman Pab., New York, all of which ,95 are. ~ ,)rporated sor all puiL :!

Overview [0032] The present invention is directed to methods and compositions for acquirinLi nucleotide sequence information of target seqLzences (also referred to herein as "target polynucleotides' ) using adaptors interspersed in target poly-riucleotides.
The sequence 200 information can be new, e.g. sequencing unknown nucleic acids, resequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a poiynucleotide. Such adaptors are referred to herein as "interspersed adaptors", and niay serve as platfomis for interrogating adjacent sequences using various sequencing chemistries, such as those that 205 identify nucleotides by primer extension probe ligation, and the like.
That is one unique component of some embodiments of the invention is the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both "upstream" and "downstream" of the adaptor, sequence information of entire target sequences may be accomplished.
210 100331 Accordingly, without limitation, the inventions can generally be described as follows (it should be noted that genomic DNA is used as an example herein, but is not meant to be limiting). Genomic DNA from any organism is isolated and fragmented into target sequences using standard techniques. A first adaptor is ligated to one terminus of the target sequence. The adaptor preferably comprises a Type IIs restriction endonuclease site, which 215 cuts outside of the recognition sequence. If the enzyme results in a "sticky" end, the overhang portion can either be filled in or removed.
[0034] In one embodiment, an enzyme is used to ligate the two ends of the linear strand comprising the adaptor and the target sequence to form a circularized nucleic acid. This may be done using a single step. Alternatively, a second adaptor can be added to the other 220 terminus of the target sequence (for example, a polyA tail), and then a bridging sequence can be hybridized to the two adaptors, followed by liiaation. In either embodiment, a circular sequence is formed.
(0035) The circular sequence is then cut with the Type Ils endonuclease, resulting in a linear strand, and the process is repeated. This results in a circular sequence with adaptors 225 interspersed at well defined locations within previously contiguous target sequences.
(ÃI0351The circularized sequences are then amplified using a rolling circle replication (RCR) reaction, to form concatemers of the original target sequence (e.g.
multimers of rn-rlomers). "I'hese ior.~Q concatemers f-rm "DNJA r-m(yb l's" ("DNI3s'"j can then opt ;;,;IIv = a ~ ir ,~ -- .. 7 230 100371 Once on the surface, using the known adaptor sequences, sequencing of the intervening tar(jet sequences is done. As is kno,~vn in the art, there are a number of techniques that can be used to detect or determine the identity of a base at a particular location in a target nucleie acid, includiDg. but not lin-tited to, the use of ternperature, competitive hybridization of perfect and irnperfect probes to the target sequence, sequencing 235 by synthesis, for exampte using single base extension techniques (sometimes referred to as :`minisequencing"), the oligonucleotide ligase amplification (OLA) reaction, rolling circle replication (RCR), alielic PCR, competitive hybridization and Invader"m tec-b.nologies.
Preferred embodiments include sequencing by hybridization with ligation, and sequencing by hybridization.

240 100381 "I'be sequence information can then be used to reconstruct sequences of larger target sequences, such as sequencing of the entire genomic DNA.
[0039] Sequencing large numbers of nucleic acids, as is necessary in applications such as I;enome analysis, epidemiological studies, and diagnostic tests, generally involves adapting sequencing technologies to high-throughput formats. However, there are drawbacks to 245 traditional high-throuohput sequencing techniques, particularly the problem of short sequence read lengths - that is, many high-throughput sequencing approaches are limited in the length and type of target polynucleotides that may be successfully sequenced. This limitation is primarily due to the number of contiguous bases that can be d.ctermined on a single frabznent in a single operation. By providing a plurality of sites in each target 250 polynucleotide or fragment firom which to conduct particular sequencing chemistries, the present invention provides a multiplicity of adjacent sequence reads. In one aspect, these adjacent reads are contiguous, thereby effectively amplifying the expected read lengths of a large class of sequencing chemistries.
100401 The present invention thus allows the determination of a longer contiguous or 25-s almost contiguous target sequence by determining the sequences on either side of adaptors.
Conr ositionslstructures o tar et polynticleotides 100411 Accordingly, the present invention provides compositions an.d methods utilizing target sequences from samples. As will be appreciated by those in the art, the sample solution may comprise any number of things, including, but not limited to, bodily fluids 260 (including, but not limited to, blood, urine, serum, lymph, saliva, anal and va~_Yinal secretions, pc.rspiration and semen) and cells of virtually any orLyanism, with mammalian samples being samples beh_,,,_T

(including, but not limited to, air. agricultural, water and soil samples);
biological warfare agent samples; research samples (i.e. in the case of nucleic acids, the sample may be the 265 products of an amplification reaction, including both target and signal amplification, such as PCR amplification reactions; purified samples, such as purified genomic DNA, RNA
preparations, raw samples (bacteria, virus, genomic DNA, etc.); as will be appreciated by those in. the art, virtually any experimental manipulation may have been done on the samples.

270 [0042] In general. cells from the target organisrri (animal, avi.an, mammalian. etc.) are used. When genomic DNA is ased, the amount of genomic DNA required for constructing arrays of the invention can vary widely. ln one aspect. for mammalian-sized genomes, fragments are generated from at least about 10 genome-equivalents of DNA: and in another aspect, fragments are generated from at least about 3 )0 genome-equivalents of DNA; and in 275 another aspect, fragments are generated from at least about 60 genome-equivalents of DNA.
[0043] The target sequences or target polynucleotides are nucleic acids. By "nucleic acid"
or "oligouucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below (for example in the 280 construction of primers and probes such as label probes), nucfeic acid analogs are included that may have alternate backbones, carnprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; l..etsint",er, J.
Org. Chem.
35:3800 (1970); Sprinzl et al., Eur. J. f3iochem. 81:579 (1977): I.-,etsinger et al.. Nuc1. Acids Res. 14:3457 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J.
Am. Chem.
285 Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat.
No.
5,644,448), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues:
A
Practical Approach, Oxford University Press), and peptide nucleic acid backbones and 290 linkages (see Egholm, J. Am. Chem. Soc. 114: 1 895 (1992); Meier et al., Chem. lnt. Ed.
Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (I996). all of which are incorporated by reference). Other analog nucleic acids include those with bicyclic structurc;s including locked nucleic acids, Koshkin et al., J.
Am. Chem. Soc.
120:13252 3 (1998), positive backbones (Denpcy et al., proc, Nati. Acad. Sci.
USA 92:6097 295 (1905 n i4 ' ~ -noncs `1;.S. 1'at.'N' ? ,684. ~ _ _ ~t?, 5m" .141 aiid >0e; ~~"
. , . ~'~ .. _ . ~ , = ; 'r . , ~~ ;~
f s _e.~..,.. _ . 9 al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside &
Nucleotide I 3:1597 (1994), Chapters 2 and 3, ASC Syrnposium Series 580, "Carbohydrate Modifications in Antisense Researcb.", Ed. Y. S. Saa.-hui and F. Dan Cook: Mesmaeker et al.,l3ioorl;anic &
300 Medicinal Chem. I.ett. 4:395 (1994); Jeffs et al., J. 1=3iomolecular N1LIR
34:17 (1994);
Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, includint', those described in U.S. Pat. Nos. 5,235,033 and 5,034,505, and Chapters 6 and 7, ASC Syrnposium Series 580, "Carbohydrate Modifications in Antisense Research'", Ed. Y. S. Sanghui and P.
Dan Cook.
Nucleic acids containing one or more carbocyclic sugars are also included within the 305 definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169 176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 paoe 35.
All of these references are hereby expressly incorporated by ref'erence. 1-hese modifications of'the ribose-phosphate backbone may be done to increase the stability and balf'-life of such molecules in physiololgical environments. For example, PNA:DNA hybrids can exhibit 310 higher stability and thus may be used in some embodiments.
100441 The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acids may be DNA, both genomic and eDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including 315 uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
100451 The term "target sequence" or "target nucleic acid" or grammatical equivalents herein meaiis a nucleic acid sequence on a single strand of nucleic acid. The target sequel7ce niay be a portion of a gene, a regulatory seqLzence, genomic DNA. cDNA, RNA
including 320 mRNA and rRNA, or others. As is outlined herein, the target sequence may be a target sequence from a sample, or a secondary target such as a product of an amplification reaction, etc. It may be any length.
100461 As is outlined more fully below, probes are made to hy'bridize to target sequences to determine the presence or absence of the target sequence in a sample.
Generally speaking, 325 this term will be understood by those skilled in the art. The target sequence may also be comprised of different target dornains; for example, a first target domain of the sample target sequence may hybridize to a capture probe and a second target domain may hybridize to a label probe, etc. The tar~.~et domains may be adjacent or separated as indicated. Unless the terms "first" and "secGT`.ed" aro not m `_1t to Ã;c.1'~- : an orientation of the or. FC r exam, pIt.

a 5'-3' orientation of the complementarv target seqnence, the first target domain mav be located either 5' to the second domain, or 3' to the second domain.
100471 In one embodiment, genomic DNA, particular human genomic DNA, is used.
Genomic DNA is obtained using conventional techniques, for example, as disclosed in 335 Sambrook et al., supra, 1999; Current Protocols in Molecular Biology, Ausubel et al., eds.(John Wiley and Sons, Inc., NY, 1999). or the like, Important factors for isolating genomic DNA include the f'ollowing: 1) the DNA is free of DNA processing enzymes and contaminating salts; 2) the entire genome is equally represented; and 3) the DNA fragments are between about 5,000 and 100,000 bp in length.
340 100481 In many cases, no digestion of't.be extracted DNA is required because shear forces created during lysis and extraction will l;enerate fragments in the desired range. In another embodiment, shorter fragments (1-5 kb) can be generated by enzymatic fragmentation using restriction endonucleases. In one embodiment, 10-100 genorne-equivalents of DNA ensure that the population of 1:ragments covers the entire genome. In some cases, it is advantageous 345 to provide carrier DNA, e.g. unrelated circular synthetic double- stranded DNA, to be mixed and used with the sample DNA whenever only small amounts of sample DNA are available and there is danger of losses through nonspecific binding, e.g. to container walls and the like. In one embodiment, the DNA is denatured after fragmentation to produce single stranded fragments.
350 [00491 Target polynucleotides may be generated from a source nucleic acid, such as genomic DNA, by fragmentation to produce fragnients of a specific size; in one embodiment, the fragments are 50 to 600 nucleotides in length. In another erxzbodiment, the fragrnents are 300 to 600 or 200 to 2000 nucleotides in length. In yet anotfier embodiment, the fragments are 10-100, 50-100, 50-300, 100-200. 200-300, 50-400, 100-400, 200-400, 355 400-500, 400-600, 500-600, 50-1000, 100-1000. 200-1000, 300-1000, 400-1000, 500-1000.
600-1000, 700-1000, 7100-900, 700-800, 800-1000, 900-1000, 1500-2000, 1750-2000, and 50-2000 nucleotides in length. These fragments may in turn be circularized for use in an RCR reaction or in other biochemical processes, such as the insertion of additional adaptors.
100501 PoIynucleotides of the invention have interspersed adaptors that permit acquisition 360 of sequence information from multiple sites, either consecutively or simultaneously.
Interspersed adaptors are oligonucleotides that are inserted at spaced locations within the interior region of a target polynucleotide. In one aspect, "interior" in reference to a target polvnr:cleotide means a site internal to a target polvnuclcotide prior to processing; such as 1l circularization and cleavage, that may introduce sequence inversions, or like 365 transformations. which disrupt the ordering of nucleotides within a tarLet po[ynucleotide.
[0051] In one aspect, as is more fully outlined below, interspersed adaptors are inserted at intervals within a conti-uous region of a target polynucleotide. In some cases, such intervals have predetermined lengths, which may or may not be eclual. In other cases, the spacing between interspersed adaptors may be known only to an accuracy of from one to a few 370 nucleotides (e.g. from 1 to 15), or froin one to a few tens of nucleotides (e.g. from 10 to 40), or from one to a few hundreds of nucleotides (e.g. from 100 to 200).
Preferably, the ordering and number of interspersed adaptors within each target polynucleotide is known.
In some aspects of the invention, interspersed adaptors are used together with adaptors that are attached to the ends of target polynucleotides.
375 [0052) In one aspect, the invention provides target polynucleotides in the form of concatemers which contain multiple copies (e.g. "monomers") of a target polynucleotide or a fragment of a target polynucleotide. DNA concatemers under con-ventional conditions (a conventional DNA buffer, e.g. TE, SSC, SSPE, or the like, at room temperature) form random coils that roughly fill a spherical volume in solution having a diameter of from about M 100 to 300 nm, which depends on the size of the DNA and buffer conditions, in a manner well known in the art, e.g. Edvinsson, "On the size and shape of polymers and polymer complexes," Dissertation 696 (University of Uppsala, 2002).
[0053] One measure of the size of a random coil polymer, such as single stranded DNA, is a root mean square of the end-to-end distance, which is roughly a measure of the diameter of 385 the randomly coiled structure. Such diameter, referred to herein as a "random coil diazrreter," can be measured by light scatter, using instruments, such as a Zetasizer Nano Svstem (Malvern Instruments. UK), or like instrument. Addition.al. size measures of macromolecular structures of the invention include molecular weight, e.g. in Daltons, and total polymer length, hicl"i in the case of a branched polymer is the sum of the lengths of all 390 its branches.
[0054[ Upon attachment to a surface, depending on the attachment chemistry, density of linkages, the nature of the surface, and the like, single stranded polynucleotides fill a flattened spheroidal volume that on average is bounded by a region which is approximately equivalent to the diameter of a concatemer in random coil contiguration.
Preserving the 395 compact form of the macromolecular structure on the surface allows a more intense signal to br- pn~tc:d by p-=*c.o, fluorÃ:scentlv labwled oiigortuc'" :~~ 4 t `-mally directed to , . _ [00551 In some embodinients, classes of polvnucleotides may be created bv providing adaptors having different anchor probe binding sites. This tvpe of "clusterin~" allows for 400 increased efficiency in obtaining sequence information of the polvnucleotides.
A1etlt ds a ra tnentation 100561 Effective m.appinig strategies are needed for sequencing applications such as seqcten.e.ing complex diploid genomes, de novo secluencing, and sequencingmix.tures of genomes. ln one ei-nbodiment, hierarchical fragrnentation procedures are provided to 405 identify haplotype information and assemble parental chromosomes for diploid genomes.
Such procedures may also be applied to predictino, protein alleles and to mapping short reads to the correct positions within a genome. Another use for such methods is the correct assignment of a mutation in a gene family which occurs within -100 bases of DNA
sequence shared between multiple genes.
410 f00571 Fi;. (1 C-D) illustrates one aspect of the invention, in which source nucleic acid (1600) (wliich may be, or contain, a single or several target polvnucleotides) is treated (1601) to form single stranded fragments (1602), preferably in the range of from 50 to 600 nucleotides, and more preferably in the range of froni 300 to 600 nucleotides, which are then ligated to adaptor oliQonucleotides (1604) to form a population of adaptor-fragment 415 conjugates (1606). Adaptor (1604) is usually an initial adaptor, which need not be "interspersed" in the sense that it separates two sequences which were contiguous in the oriainal sequence. Source nucleic acid (1600) may be genomic DNA extracted from a sample using conventional techniques, or a cDNA or genomic library produced by conventional techniques, or synthetic DNA, or the like. Treatment (I601) usually entails 420 fragmentation by a coraventional technique, such as chemical fragmentation, enzymatic fragmentation, or mechanical fragmentation, followed by denaturation to produce single stranded DNA fragments.
[00581 In generating fragments in either stage, fragments may be derived from either an entire genome or from a selected subset of a genome. Many techniques are available for 425 isolating or enriching f'ragments from a subset of a genome, as exemplified by the following references, which are incorporated in their entirety by reference: Kandpal et al (1990), Nucleic Acids Research, 18: 1'189-1795; Ca)low et al, U.S. patent publication 2005i0019776; Zabeau et al, U.S. patent 6,045.994; Deugau et al, L.S. patent 5,508,169;
Sibsonõ U.S. patent 5,728.5324; Guilfovle, et al, U.S. patent 5,994,068; Jones et al, U.S. patent 430 publication 2405/0142577; Gullberp- et al, U.S. patent publication 2005I0037356; Matsuzaki et al, U.S. patent ptiblication 2004!0067493; and the like.
[00591 In one embodiment, shear forces during lysis and extraction of genorraic DNA
generate fragments in a desired range. Also encompassed bNr the invention are metbods of fragmentation utilizing restriction endonucleases.
435 100601 In a preferred embodiment, particularly for mammalian-sized genomes, fragmentation is carried out in at least two stat;es, a first stage to generate a population of fraf;ments in a size range of from about 100 kilobases (Kb) to about 250 kilobases, and a second stage, applied separately to each 100-250 Kb fragment, to generate fragments in the size ranf;e of from about 50 to 600 nucleotides, and more preferably in the range of from 44o about 300 to 600 nucleotides, for generating coneatemers for a random array. In some aspects of the invention, the first stage of fraurmentation may also be employed to select a predetermined subset of such fragments, e.g. fragments containing genes that encode proteins of a signal transduction pathway, and the lilCe.
[00611 In one embodiment, the sample genomic DNA. is fragmented using techniques 445 outlined in US Ser. No. 11/45I,692, hereby incorporated by reference in its entirety. In this aspect, genomic DNA is isolated as 30-300 kb sized firagments. Throu ;.b proper dilution, a small subset of these fragments is, at random, placed in discreet -vvells of multi-well plates or similar accessories. For example a plate with 96, 384 or 1536 wells can be used for these fragment subsets. An optimal way to create these DNA aliquots is to isolate the DNA with a 450 method that naturally fragments to hioh molecular weight forms, dilute to 10-30 genome equivalents after q.uantitation, and then split the entire preparation into 384 wells. This provides representation of a] l genon-iic sequences, and performing DNA
isolation on 10-30 cells with 100 % recovery- efficiency assures that all chromosomal regions are represented with the same coverage. By providing aliquots in this method, the probability of placin~ two 455 overlapping fragments from the same region of a chromosome into the same plate well is minimized. For diploid genomes represented with lOx coverage, there are 20 overlapping fragments on average to separate into distinct wells. If this sample is distributed over a 384 well plate, then each well contains, on average, 1.562 fragments. Bv forminf-, 384 fractions in a standard 384-well plate. there is onlv about a 1/400 chance that two overlapping 460 fragments may end up in the same well. Even if some matching fragments are placed in the same well, the other overlapping fragments f:roni each chromosomal region provide the tinique mapping i.uformation.

(0062] In one embodinaent, the prepared groups of Ion.g fragments are further cut to the final fragment size of about 300 to 600 bases. To obtain sufficient (e.g., IOx) coverage of 465 each fragment in a group, the DNA in each well may be amplified before final cutting using well-developed whole genome amplification methods.
[00631 All short fragments from one well may then be arraved and sequeiaced on one separate tinit array or in one section of a larger continuous matrix. A
composite array of 384 unit arravs is ideal for parallel anafvsis of these groups of fragments. In the assembly of long 470 sequences representing parental chromosomes, the algorithm may use the critical information that short fragments detected in one unit array belong to a limited nunlber of Ionger continuous segments each representing a discreet portion of one chromosome. In almost all cases the homologous chromosomal segments may be analyzed on different unit arrays. Long (-100 Kb) continuous initial segments form a tailing pattern and provide 475 sufficient mapping information to assemble each parental chromosome separately as depicted below by relying on about 100 polymorphic sites per 100 kb of DNA. In the following example dots represent 100-1000 consecutive bases that are identical in corresponding segments.
Well 3 ......T.... ....C.... ,.....C...G.......... A..........
480 Well 20 ....C........T.......... T ...A...... .G.........C...
Well 157 .......T. .A...... ..G... ...C........ A...C.
Well 258 ...C.... ......C...G.......... A......... T........G...T....
Wells 3 and 258 assemble chro:nosorne I of Parent I:...T........ C..........
C...G....... A..... .....T........ G... T
Wells 20 and 157 assemble chroÃnosome 1 of Parent 2:...C........ T......
..T...A.......... G... ...C........A...C...

100641 In one embodiment, amplification of the single targets obtained in the chromosomal separation procedure is accomplished using methods known in the art for whole genome amplification. In a preferred embodiment, methods that produce 10-100 fold amplification are used. In one embodiment, these procedures do not discriminate in terms of the 490 sequences that are to be amplified but instead amplify all sequences within a sample. Such a procedure does not require intact amplification of entire 100 kb fragments, and shorter fragments, such as fragments from 1-10 kb, can be used.

C mbasition/structure of cnlerspersed adaptors 1(}0651 In one aspect, interspersed adaptors are inserted at intervals within a contiguous 495 region of a target poIvnuc-leotide. Interspersed adaptors may vary widely in length, which depends in part on the number and type of functional elements desired. Such functional elements include, but are not limited to, anchor sequences, sequences complementary to "1t a :':. 'it " `l'-~ .-~~t- .~.-, i =e>r3 . ...i.
, .,.. _ . i :.-,_ . . ., . .-, .

structure sequences, sequences for attachment/hybridization of label Iarobes.
500 functionalization sequences, primer binding sites, recognition sites for nucleases, such as nicking enzymes, restriction endoniicleases, and the like.
(0066] In one embodiment, the adaptors comprise a restriction endonucle.ase recognition site as k-nown in the art. In one embodiment, such recognition sites can be for nicking enzymes.
505 [0067] In one embodiment, the restriction endonuclease site is a Type IIs restriction endonuclease site. Type-IIs endonucleases are generally commercially available and are well known in the art. Like their Type-II counterparts, Type-Iis endonucleases recognize specific sequences of nucleotide base pairs within a double stranded polynucleotide sequence. Upon recognizing that sequence, the endonuclease will cleave the polvnucleotide 510 sequence, generally leaving an overhang of one strand of the sequence, or "sticky end."
Type-Ils endonucleases also generally cleave outside of their recognitior-sites; the distance may be anywhere from 2 to 20 nucleotides away from the recognition site.
Because the cleavage occurs within an ambiguous portion of the polynucleotide seclue ce, it permits the capturing of the ambiguous sequence up to the cleavage site, under the methods of the 515 present invention. Usually, type IIs restriction endonucleases are selected that have cleavage sites separated from their recognition sites by at least six nucleotides (i.e.
the number of nucleotides between the end of the recognition site and the closest cleavage point).
Exemplary type IIs restriction endonucleases include, but are not limited to, Eco57M I, Mme I, Acu I, Bpm 1, BceA I, Bbv I. BeiV I. BpuE I, BseM 11, BseR 1, Bsg 1, BsmF
I, BtgZ I, Eci 520 1, EcoP15 I, Eco57M I, Fok I, IIga I, I-Iph I, Mbo 11, Mnl I, SfaN I, TspDT I, TspDW I, Taq 11, and the like.
100681 In some embodiments, each adaptor comprises the same I`ype IIs restriction endonuclease site. In alternative embodiments, different adaptors comprise different sites.
t0069] In one embodiment, one or more of the adaptors comprise anchor probe 525 hybridization sites. As is outlined below, anchor probes are u.sed in sequencing reactions, and can take a variety of forrns. In general, at least one end of t1ae anchor probe hybridization site is at the junction between the target sequence and the adaptor; that is, sequencing reactions generally rely on hybridization of the anchor probe directly adjacent to detection positions of the target sequence. The anchor or primer may be selected or designed 530 to be or to have one to about ten or more, preferably one to four bases, shifted left or ripht a:~ap*, As Ã~scd'i ters to a 1 r _quence [0070] In manv embodiments, sequencing reactions can be run off both ends of the anchor probes; thLIs, in some embodiments. the anchor probe hybridization site comprises the entire 535 adaptor sequence. Alternatively, there may be t-wo anchor probe hybridization sites within each adaptor; one adjacent or close to the 3' end of the target sequence and one adjacent or close to the 5' end. As will be appreciated by those in the art, depending on the length of the anchor probes and the length of the adaptor, two anchor probe hybridization sites may overlap within the adaptor, they may be directly adjacent, or they may be separated by 540 intervening sequences. The length of the anchor probe hybridization sequence will vary depertdin~,~ on the conditions of the assay.
100711 In one embodiment, one or more of the adaptors comprise a primer binding sequence. As is known in the art, polymerases generally require a single stranded template (the concatemers, for example) with a portion of double stranded nucleic acid.
Essentially, 545 any sequence can serve as a primer binding sequence, to bind a primer, as any double stranded sequence will be recognized by the polymerase. In general, the primer binding sequence is from about 3 to about 30 nucfeotides in length, with from about 15 to about 25 being preferred. Primer oligonucleotides are usually 6 to 25 bases in length.
As will be appreciated by those in the art, the primer binding sequence can be contained within any of 550 the other adaptor sequences.
[0072) In one embodiment, one or more of the adaptors comprise a capture probe recognition sequence. As is more fully outlined below, one embodiment of the invention utilizes capture probes on the surface of a substrate to immobilize the DNBs.
ln this embodiment, the adaptors comprise a domain sufficiently complementary to one or more 555 capture probes to allow hybridization =fthe domain and the capture probe, resulting in immobilization of the DNBs on the surface.
(0073) In one embodiauent, one or more of the adaptors comprise a secondary structure sequence. For exarnple, palindromic sequences in a plurality of adaptors within the concatemer results in hybridization between adaptors (e.g. intramolecular interactions 560 between copies in the concatemer) thus "tightening" the three dimensional strticture of the DNA nanoball ("DN1=3s'"}. `1'hese palindromic sequence units can be 5, 6, 7, 8, 9, 10 or more nucleatides in length and of various sequences, such as sequences chosen to provide a specific melting temperature. For example, a palindrome AAAA~AATTTT S'T`I' w-ill provide a 14 bases dsDNA hybrid between neighboring any two unit replicas in the form of:
565 A iAAAA",:11,T`;,7'r;"1-.T

L.

100741 In one embodiment, the adaptors cornprise iabel probe binding sequences. In some embodiments, for example for detection of particLilar sequences rather than sequencing reactions. label probes can be added to the concatemers to detect particular sequences. Label 570 probes will hvbridize to the label probe binding sequence and comprise at least one detectable label, as is outlined herein. For e?cample, detection of the presence of infectious a[zents such as bacteria or viruses can be done in this manner.
[0075] In one embodiment, the adaptors comprise tagging sequences. In this embodiment, tagging sequences may be used to pull out or purify circularized target sequences.
575 concatemers, etc. In some embodiments, tagging sequences may inciude unique nucleic acid sequences that can be utilized to identify the origin of target sequences in mixtures of tagged samples, or can include components of ligand binding pairs, such as biotin/streptavidin, etc.
100761 In one aspect, interspersed adaptors each have a length in the range of from 8 to 60 nucleotides; in another aspect, they have alength in the range of from 8 to 32 nuclcotides: in 580 another aspect, they have a length in a range selected from about 4 to about 400 nucleotides;
from about 10 to about 100 nucleotides, from about 400 to about 4000 nucleotides, from about 10 to about 80 nucleotides, from about 20 to about 70 nucleotides, froni about 30 to about 60 nucleotides, and from about 4 to about 10 nucleotides. Embodiments utilizing adaptors with a total length from about 20 to about 30 bases find particular use in several 585 embodiments.
[0077] The number of interspersed adaptors inserted into target polynucleotides may vary widely and depends on a number of factors, including the sequencing/genotyping chemistry being used (and its read-length capacity), the partieular length of the cleavage site of a particular Type IIs site, the number of nucleotides desired to be identified within each target 590 polynucleotide, whether amplification steps are employed between insertions, and the like.
100781 In one aspect, a plurality of interspersed adaptors are inserted at sites in a contiguous segment of a target polynucleotide; this may include two, three, four or inore interspersed adaptors that are inserted at sites in a contiguous segment of a target polynuelecrtide. Alte-rnatively, the number of interspersed adaptors inserted into a target 595 polynucieotide ranges from 2 to 10. from 2 to 4; from 3 to 6; from 3 to 4;
and from 4 to 6.
In another aspect, interspersed adaptors may be inserted in one or both polynucleotide segments of a longer polvnucleotide, e.g., 0.4-4 Kb in length, that have been ligated together directly or indirectly in. a circularization operation (referred to herein as a-`mate-pair"). In or_:; such. l -r1cleotide setl-merts may be 4-400 (pre:ferabiy 10-100) base.<

600 100791 It should also be noted that in general, the first adaptor attached to a target sequence is not "interspersed" or "inserted". That is, the first adaptor is generally attached to one terminus of the fragmented target sequence, and the subsequent adaptors are interspersed rvithin a contiguous target sequence.
(0080] In one aspect, each member of a group of target polynueleotides has an adaptor 605 -with an identical anchor probe binding site and type Ils recognition site attached to a DNA
fragment from source nu.cleic acid. In another embodiment, classes of polynucleotides may be created by providing adaptors having different anchor probe binding sites.
[0081] In one aspec-t, adaptors are inserted at intervals within a contiguous region of a target polynucleotide in which the intervals have pre-determined lengths.
These pre-61tt determined lengths may or may not be equal. In some embodiments the length ofth.e intervals are known to an accuracy of about 1 to 200 nucleotides, in other embodiments from about 1-15, 10-40 and ] 00-200 nucleotides.
[00821 Interspersed adaptors may in accordance with the invention be single or double stranded.
615 100831 In one aspect, adaptors include palindromic sequences, which foster intramolecular interactions within the target polynucleotide, resulting in a"nano-ball".

Methods for insertirag aplura-lrty nFadaEtors [0084] One aspect of the invention provides a method for producing a target polynucleotide having interspersed adaptors, as illustrated diagrammatically in Figs. (IA-620 IB). In this method, taraet polynueleotide (1002) is combiDed with adaptor (1000), which may or may not be an interspersed adaptor, to form (1004) circle (1005), which may be either single stranded or double stranded. The target polynucleotide is generally obtained by fragmentation of a larger piece of DNA, such as chromosomal or other genomic DNA.
[0085] If double stranded DNA is used, then the ends of the fragments may be prepared for 625 circularization by "polishing" and optional ligation of adaptors using conventional techniques, such as employed in conventional shotgun sequencing, e.g. Bankier, Methods Mol. Biol., 167: 89-100 (2001); Roe. Methods Mol. Biol., 255: 1?1-185 (2004);
and the like.
100861 In order to generate the iiext site for inserting a second interspersed adaptor. circle (1005) is t}rpically rendered double stranded, at least temporarily. Adaptor (1000)is 530 designed in this aspect of the invention to include a recognition site oI_a type lis restriction endonuclcasc, which is oriented so that its cleavage site (1006) is int_,.rior to the target circle (I005). In a preferred embodiment, the method of inserting interspersed adaptors employs type IIs restriction endonucleases that leave 3' protruding strands after cleavage.
635 For less precise insertion, a nicking enzyme may be used, or one strand of the tirst adaptor may be disabled from ligation, thus creating a nick that can be, translated at an approximate distance and tised to initiate polynucleotide cutting.
100871 After the polynucleotide is cleaved, interspersed adaptor (101(}) is ligated into place using conventional techniques to produce open circle (101.2) containing two adaptors, 640 which is then closed (1016) by ligation. The process is then repeated (101$): cleaving, inserting, and closing, until a desired number of interspersed adaptors, such as three, are inserted (1026) into target polynucleotide (1002). as shown in Fig. 3B. The final circle (1024) containing the interspersed adaptors may then be processed in a number of ways to obtain sequence information at sites in the target polynucleotide adjacent to at least one 645 boundary of each interspersed adaptor.
100881 Typically, sequences of a target polynucleotide are analyzed at or adjacent to one or both of the boundaries (e.g. 1021) between each interspersed adaptor and the target polynucleotide. In one aspect, final circle (1024), or a segment of it, may be amplified to generate an amplicon that is analyzed by a selected sequencing chemistry, such as one based 650 on ligation or sequencing-by-synthesis. In one aspect, the first and last iliterspersed adaptors niay be selected so that the region of final circle (1024) containing the interspersed adaptors can be cleaved (1038) from the circle, after which adaptors are ligated (1040) for amplification by polymerase chain reaction (PCR). Cleavage of the circle may be performed on one or two sites outside of adaptors I and 3. In another aspect, final circle (1024) may be 655 used directly to generate amplicons by rollin- circle replication (RCR), as described more fully below.
140891 For applications in which many different target polynucleotides are analyzed in parallel, target polynucleotides having interspersed adaptors may be amplified using RCR or emulsion PCR as shflw-n in Figs. (1 C-1D) and Figs. {] E-1 G), respectively.
660 100901 In emulsion PCR, a mixture of fragments may be arnplified, e.g. as disclosed by Margtilies et al, Nature, 437: 376-380 (2005); Shendure et al (2005). Science, 309: 1728-1732; Berka et al, L.S. patent publication 2005s[I07951 0; Church et al, PC"I"
publicatiQn WO
2005/082098; tiQbile et al, U.S. patent publication 2005/0?21i 264; Griffiths et al. U.S. patent 6,489,103: "T'illett et al, PCT publication Vv'O 03!106678: Kojizna et al, Nucleic Acids 565 (17}: el-50 (2005): Dreszman et al, I' L M d'. A--ade Sci., IM 8817-8822 'mal, B . Nvch et al, Biomacromolecrtles. 6: 1824-1828 (2005); Li et al. Nature Methods, 3: 95-97 (2006); and the like, which are incorporated herein by reference in their entirety for all purposes.
100911 Briefly, as illustrated in Fig. (lE), after isolation of DNA circles (1500) comprising 670 target polynucleotides with interspersed adaptors, the adaptors are excised, e.g. as shown in Fig. lA (1038), to form a population of excised sequences, which are then iil;ated to adaptors (1503). The adaptored sequences are combined in a water-oil emLiision (1505) with primers specific for an adaptor ligated to one end of cxcised sequences, beads having attached primers specific for an adaptor iigated to the other end of excised se:quenc-es, and a 675 DNA polymtrase. Conditions are selected that permit a substantial number (e.g. greater than 15-20 percent) of aqueous bubbles (1508) in oil (1506) to contain a single adaptored sequence (1510) and at least one bead (1512). The aqueous phase in bubbles (1508) otherwise contain a conventional reaction mixture for conduction PCR, which results in beads (1518) each having a clonal population of a distinct adaptored sequence attached.
680 100921 In one aspect of the invention, the introduction of multiple interspersed adaptors into a single genomic fragment proceeds through a series of steps involving 1) ligation of an initial adaptor harboring a binding site for a Ils restriction enzyme and closing the DNA
circle, followed by 2) primer extension and selective restriction cutting of the genomic sequence to reopen the circie; and 3) Iigation. of second adaptor and closing the DNA circle.
685 Steps 2 and 3 are then repeated to incorporate a third adaptor into the genomic sequence (Figs. 2B and 2C). The second adaptor may utilize the same restriction site as the first adaptor to minimize cutting genomic segments at an internal site of the genolnic DNA. In one embodiment, controlled cleavage using the recognition site of the second adaptor and not of the first adaptor is accomplished by blocking the cleavage at the l~irst adaptor 690 restriction site using techniques known in the art, such as by methylating the first restriction site prior to cutting at the second site.
100931 Adaptors with different binding sites may be used with two aliquots of a sample to prevent exclusion of certain genomic fragments. In one embodiment, a part of tlie sequence of the tinal adaptor is used as an RCR priming site and another part of the adaptor is used as 69 5 a binding site for an anchor oligonucleotide attached to a glass surface.
[00941 In one aspect of the invention, a method for inserting adaptors into a genomic fragment begins with ligation of a first adaptor followed by circle forination. Genomic fragments of 100 to 300 (or 300-600) bases in length may be prepared by D NAse ~T`x x~I rlt"1` ~t that ;:; Gr'7f:'ti 5-p:"in"'w pktC}Sp~"..:_ 01i r.'S s`,,.
.t 3 f 1 . . ._ ._ _ .. ~, ., . , DNA by heating (denaturation) and rapid cooling. Since the DNA is of high complexity, the localized concentration of the complementary sequence for any fragment may be negligible, thus allowing sufficient time to perform subsequent procedures when the DNA is mostly in the single stranded state. Tbe use of ssDNA significantly simplifies circle formation because of 705 the distinct polarity of 5' and 3' ends of each ssDNA fragment. The first stage is ligation of adaptor sequences to the ends of each single stranded genomic fragment. Since all possible sequence combinations may be represented in the genotnic DNA, an adaptor can be ligated to one end w-ith the aid of a bridging template molecule that is synthesized with all possible sequences (Fig. 2B). Since these oligonucleotides may be of relatively high concentration 710 compared to the genomic DNA, the o[igonucleotide that is complementary to the end of the genomic fragment (or a complement with mismatches) may hybridize. A bridge is thus formed at the ligation site to allow ligation of the 5-prime end of the single stranded genomic fragment to the adaptor. In one embodiment, this structural arrangement does not allow ligation of the adaptor to the 3-prime end of the fragment.
715 [0095] In Fig. 2B, another exemplary method is illustrated for incorporating multiple interspersed adaptors into DNA circles. Such method comprises the steps of: 1.
Ligation of adaptors (230) to the 5' and 3' end of single stranded DNA (232) (the adaptors having degenerate (6-9 bases) bridge templates (234)) followed by ligation of the adaptors via a 3-base overhangs (236); 2. Extension (238) from the adaptor oligonucleoiide with a 720 polymerase to create double stranded DNA for type IIs restriction enzyme cutting; 3. A cut (242) at 12-16 bases downstream of the type lls recognition site (240) opens the circle; 4.
FIeating results in loss of Dew strands (243); and 5. The fragment is ready for introduction of another adaptor (230) and closing the circle again.
100961 Capture of the 3' eiid into the circle requires the use of an oligonucleotide teinplate 72S that again is prepared with degenerate bases so that a bridge structure is formed over the ligation site. The second adaptor section at the 3' end of the genomic fragment is used to close the circle with a 3-base overhang that is complementary to the end of the adaptor that bound at the 5' end. By performing the attachment of this adaptor segment at a temperature that favors hybridization of the template bridge (but not the 3 base overhang), the excess 730 bridge molecule can be removed by buffer exchange since the genomic/adaptor molecule is attached to a solid support. A 3-base overhang is sufficient for circle formation but would not be favored until the temperature was decreased. I-he use of two bridging oli~~~rt.cl- _ 4de,~ de4-nerate bases can artafac} _._.. l~~, <~=: d~ve.rse DNA. ln L

735 oligonucleotides attach independently of each other to ensure freedom of the degenerate oligonucleotides to bind to their coinplerrmentary sequences. Both of the adaptor components ma~s be li~ated to the respective DNA ends in the same li~;ation reaction and ligation artifacts can be further prevented by designing bridging template oligonucleotides with blocked ends.
[00971 The incorporation of a capture mechanism such as biotirVstreptavidin onto the 74o non-circle adaptor strand can be used in a down-stream cleanup processes.
In such an embodiment, since both unligated and ?i:gated biotynilated adaptors are present, the un-Iigated excess adaptor can be removed by size selection of adaptor-genomic fragments that are -200 bases in length. The adaptor-genomic fragments can then be attached to streptavidin. coated beads for subsequent cleaning procedures. Another option is to use beads 745 with a capture oligonucleotide (possibly incorporating PNA or LNA) complementary to a portion of one ligated adaptor. Beads with a pre-assembled left side of the first adaptor/template may be used to further simplify the process.
100981 In Fig. 2C, another exemp?ary method for incorporating interspersed adaptors is illustrated. The method comprises the following steps: (1) Ligate two adaptor segments 750 (250 and 252) to single stranded DNA fragments (254) using template oligonueleotides (the double stranded segment of 250 may be about 10 bases long, and the double stranded segment of 252 may be 8 -10 bases long) containing degenerate bases (for example, segments 256 and 258 show the use of 7 degenerate bases, but 8 degenerate bases could also be used). Both ends of template oligonucleotides (250 and 256) are blocked from ligation 755 with dideoxy termination on the 3' ends and either 014-group or biotin on the 5' ends. The adaptor/template hybrids are used at very hil;h cone entrati ons such as 1 p.M
and are in 1000-folds excess concentrations over genomic DNA. (2) DNA is collected on streptavidin support (260) via the biotin on the 5' end of'the 3' adaptor (250). Excess free 5' adaptors are removed with the supernatant. (3) DNA is released from the streptavidin support by 760 elevated temperature and the supernatani is collected. (4) DNA is recaptured to a solid support usin_2 a[oriz; capture oligonacleotide (262) with 3' ertd blocked by dideoxy terrnination. 'I`he oligonLacleotide may be in the form of a peptide nucleic acid (I'N A ) to provide tight binding of the DNA to the solid support to facilitate removal of excess free adaptors in subsequent procedures. Capture oligonucleotide (262) can be extended by-765 addition of 1-1 0 degenerate bases at the 5' end (264) for binding the genomic portion to increase stability. (5) 'I'h.e bridge template (266, which may be 1.4-1S bases long) is used to b_rim! Iy- t~,- f~nds,-f the ad.~lnt -s te,.c th~_ :- to circularize the DN A
I` will be b. the Y 0. i subsequent elongation by DNA polymerase in later steps. K.inase and lilgase are provided in 770 the reaction to phosphorylate the S` end of the 5` adaptor and the ligation of the two ends of the DNA molecule.
100991 In another exemplary capture procedure for inserting multiple adaptors, two adaptor setiments are ligated to genomic ssDNA fragments using devenerate templates (Fig. 2C).
The 3' end of the adaptor seaznent that ligates to the 5' end of the genomic DNA has a 775 blocking complement. The template for the 3' adaptor segment has biotin.
Adaptor/
templates are in very high concentration such as I p.M and have -1000x high concentration from genomic DNA. DNA is collected on a streptavidin support and the solution is removed with the excess of adaptor components. The genomic DNA is released at an elevated temperature and the DNA solution is collected. The DNA is collected again on a second 780 solid support with a long oligonucleotide (with blocked ends) complementary to the 5' end adaptor segment with removal of all other synthetic DNA. A bridging template is then added that serves also as a primer. Kinase and ligase (and polymerase) are added to close the circle and extend the primer to about 30 bases. !-?xtension is controlled by time or by presence of ddNTPs. The enzyn-tes are heat inactivated and the DNA. is then cut with a type 785 IIS restriction enzyme. The short double stranded portions are removed at elevated temperature with the circle attached to the solid support via a strong hybrid to the attached oligonucleotide. This stronger hybrid is maintained by incorporating LNA or PNA bases into the oligonucleotide. Two adaptor segments with templates for the second adaptor are then added (same design as above) no additional solid support attacliment i.s required since 790 the circle DNA will be continually associated with the solid support for further steps.
Elevated ternperatures are used to remove templates bound to the circular DNA.
This step is repeated to insert a third adaptor. If no additional adaptors are to be inserted, then no polymerase is added and after a buffer exchange the DNA is released at elevated temperatures for the RCR reaction.
795 1001001 Another exemplary method of inserting interspersed adaptors is illustrated in Fi~-. 2D. This method generates segments of target po[ynucleotide with predetermined lengths adjacent to i.ntersperse-d adaptors. The predetermined lengths are seiected by selecting and positioning type lis restriction endonucleases within the interspersed adaptors.
In one aspect of this method, each different interspersed adaptor from the initial adaptor to 80f) the penultimate adaptor has a recognition site of a different type Iis restriction endonuclease.
Dou'-?e DNA (dsI?NA> S p roduc_ av. ~ 2 6~ ; .. -epair~
.___ .. . . _.~
~~
~

to form f'ragments (271) with blunt ends. To the 3' ends of blunt end fragments (271) a single nucleotide (;273) is added, e.g. dA, using 'f`aq polymerase, or like enzyme, to produce 805 augmented fragments (272). Augmented fragments (272) are combined with interspersed adaptors (274) that have complementary nucleotide overhangs, e.g. dT, in the presence of a ligase so that multiple ligation products form, includint-f product (275) that comprises a single interspersed adaptor and a single fragment. Conditions can be adjusted to promote the circularization (276) of product (275) so that dsDNA circles (283) are formed.
C)ther sio prodticts, such as conjugates with interspcrsed adaptors at both ends or unligated fragments and adaptors, will not generally have the ability to form circles and can be removed through digestion with a single stranded exonuclease after circularization of product (275).
(00I01] dsDNA circles (283) are treated with a type I1s restriction endonuclease recognizing a site in adaptor (278) to cleave dsDNA circles (283) to leave segment (277) of S15 target polynrtcleotide (270) adjacent to adaptor (278). In this embodiment, cleavage by the type Ifs restriction endonuclease leaves 3' indented ends that are extended by a DNA
poIymerase to form blunt ends (279), after which fragment (284) is treated to add a single nucleotide to its 3' ends, as above. To fragment (284), a second interspersed adaptor (281) having complementary overhangs is ligated, and the process repeated to incorporate 820 additional interspersed adaptors. In one embodiment, each cycle of interspersed adaptor incorporation includes an amplification step of the desired product to generate sufficient material for subsequent processing steps.
1001021 In Fig. 2E, another exemplary method is illustrated for incorporating interspersed adaptors at predetermined sites in a target polynucleotide.
Fragments are 825 generated as in Fig. 2D and dsDNA circles (285) are produced that have an initial interspersed adaptor (286) containing a type IIs recognition site, as described above, that cleaves dsDNA circle (285) at a predetermined site (287) to give fragment (288) having 3' overhangs (289), which may have lengths different than two. Interspersed adaptor of fragment (288) either contains a nick (290) at the boundary of the adaptor and the fragment 830 or it contains the recognition site for a nicking endonuclease that permits the introduction f a nick {291 j at the interior of the adaptor. In either case_ fragment (:288) is treated with a DNA polymerase (292j that cazi extend the upper strand from a nick (e.g. 29I ) tc) the end of the lower strand of fragment (288) to form a fragment having a 3' overhang at one end and a blunt end at the other. rI'o this fragment is ligated an interspersed adaptor (294) that has 9 3 5 degen~-sr-4fe n=,cl,,r~tidz, <, Nhana at one end and a sir:,,le 3' na:cleotide. {c..'_ overhG,,,~-, at 95}, g. W1th fa:[ h ~ e dA to its blunt end forming fragment (296). Fragment (296) is then circularized by ligation at site (297) to form dsDNA circle (298) and other ligation products are digested, as described above. Additional cycles of this process may be carried out to incorporate 840 additional interspersed adaptors, and as above, optional steps of amplification may be added in each cvcle, or as needed.
[00103] In Fig. 2F, another method of incorporating interspersed adaptors is illustrated that provides segments of variable lengths between interspersed adaptors, `I`hat is, interspersed adaptors are incorporated in a predetertnined order, but at spacings that are not 845 precisely known. Tb.is method allows incorporation of adaptors at distances longer than those provided by known restriction enzymes. As above, dsDNA circles (2000) are prepared having an initial adaptor (2002) (that may or may not be an interspersed adaptor) containing a recognition site (2004) for a nicking enzyme. After creation of nick (2006), dsDNA circle (2000) is treated with a DNA polymerase (2008) that extends (2010) the free 3' strand and 850 displaces or degrades the strand with the free 5' end at nick site (2006).
The reaction is stopped after a predeternined interval, which is selected to be shorter than the expected time to synthesize more than a few hundred bases. Such extension may be iialted by a variety of methods, including changing reaction conditions such as temperature, salt concentration, or the like, to disable the polymerase beiiig used. 'I'bis leaves dsDNA circle with a nick or 855 other gap (2012), which is recognized and cleaved by a variety of enzymes having nuclease activities, such as DNA polytnerases, FEN-1 endonucleases, S i nuclease (201.4), and the like, which may be used alone or in combination, e.g. Lieber, BioEssays, 19:

(1997). After cleavage at nick or gap (2012), the ends of the target polynucleotide may be repaired using techniques employed in shotgun sequencing, after which target 860 polynucleotide (2000) may be cleaved (2017) to the left of adaptor (2002) using a type Iis restriction endonuclease that leaves a staggered, or sticky, end. To the blunt end, the next interspersed adaptor is attached, after which the resulting construct may be circularized tising conventional techniques for further insertions of interspersed adaptors. In one embodiment, the distances between successive interspersed adaptors, e.g.
(2002) and (2018).
865 are not known precisely and depertdon the cleaving enzyme employed, the polymerase employed, the time interval allowed for synthesis, the method of stopping syntbesis, reaction conditions, such as dNTP concentrations, and the like.
[00104] In one ernbodiment, at step (2010), nick translation can be used instead of strand In one iw F~~ p~ n::cleotide' '''016!

870 ;.-;5,', f.ir.:tt.,.

second cut on the opposite side of the adaptor (2006) to create a mate-pair structure with various lengths of two segments such as (10-50) + (30-300) bases.
1001051 In one aspect, the invention provides a method for inserting adaptors using CircL`zgase ~~'N`l to close single stranded polynucleotide circles without template. This enzyme 875 pro~~ides the ability to use adaptors as single oli~aon:ucleotides and to use only one template.
In this method, after an adaptor is ligated to the 5' end of the target polynucleotide using standard ligase such as T4 DNA ligase, the excess adaptor and template is removed.
C.ireLigase (and kinase if the adaptor is not phosphorylated at the 5 end) can. then be used to close single stranded polynucleotide circles.
sso 1001061 In one embodiment, after the initial adaptor is inserted into the polynueleotide, it may need to be released from the support to be able to form a single stranded circle. The polynucleotide can then be re-hybridized to the support; in one embodiment, this re-hybridization occurs on a capture oligonucleotide whicli is bound to the surface of the support. A primer is added together with polyrnerase after closin- the cycle for generating 885 local dsDNA and allowing the cutting with type IIS restriction enzymes:
F-NNNNN?v'NUUE.7UUULUUUU-;
GGGGGGGGGGGGGG.LIUUUUUUUUUUUUUUUUUUUUUUUUUU-5'O}t 3"Ot-I-GGGGGGGGGG....
[001071 Ligation of multiple adaptors may be prevented by starting with 5'01-1 or by having 890 long blocking template possibly in the form of a hairpin:
~-NNNNNNNUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU-Solid UUUUUUUUUUUUUUUIrUI~ UUUCir..?UUUUU-P 1-UUUUUUU-i~
where U= common base, N=degenerate base_ I'- phosphate, G= genomic or DNA of interest.
895 1001081 Once circle formation has occurred, a pritner already pre-hybridized to the adaptor is extended with a polymerase to create enough double stranded DNA for type Ils restriction enzyme cutting allowing precisc insertion of additional adaptors {Fig 9}. A
polymerase such as Klenow may be used, along with a level of ddNTF's to control extension length to about 20-30 bases.
900 1001091 Inserting two additional adaptors can in some embodiments of the invention take 2-3 hours if each enzymatic step is accomplished in less than 3 W minutes.
Sporadic errors created in. the adaptor insertion process can be tolerated because of the redundant tens of overlappinc, sequences generated for each base and because of probe-probe data that is generated on more than 100 bases of each DNA fragment that is not sttb}ected to adaptor nise.rtion.

100I101 In one exemplary method, multiple adaptors can be inserted by preparing dsDNA
circles with a 50-100 bases +25 base mate-pair at >1 Kb distance. In this method, a dsDNA
circle of a rv1-3 Kb genoinic fragment is provided with an adaptor using A/T
or blunt-end ligation. In one embodiment, the adaptor has a nicking enzyme binding site or it has one 910 Uracil or other cleavable or photo-cleavable base ana[ot-ys or one 3' end that is not ligated a-nd recogziition sites for two different IIS binding enzymes.
[001111 In one embodimerzt, the DNA is cut using a nicking enzyrne or at Uracil sites and the available 3' end is extended (or just extended if adaptor ligation has left a nick) by -75 bases with strand-displacement enzyme or nick translation enzyme; in the case of usiiig a 915 unligated 3' site, the displacement would be through the adaptor, e.g, the Ienl;th would be 75 bases plus the length of the adaptor. The available 3' end may be removed by nick translation or by DNA synthesis with strand displacement. 'l'he cut can be at a nick or at a branched structure by one of several enzymes including single stranded cutting enzymes.
This process results in a dsDNA fragment 30-110 bases next to one end of the initial 920 adaptor. The DNA can then be cut with a Type IIS restriction endonuclease that has a ]ong cutting distance. In one embodiment, the cutting distance is from 18 to 25 bases. The circle can be closed without adaptor (blunt end ligation of genomic fragments) or by directional blunt end ligation of a second adaptor. Both adaptors may be used for further insertion of additional adaptors using different or the same enzymes. If the first adaptor site is 925 methylated before insertion of the second adaptor the second adaptor can use the same restriction site positioned at the proper distance from the adaptor end to obtain cuttinc, at the specific position in the genomic DNA.

il'let{iacls of circularizatian 1001121 Various standard DNA circle formation procedures may be used. One example is 930 blunt end ligation of the adaptor. A problem witli this approach is orientation and ligation of multiple incorporated adaptors. One strand of the cassette may have both the 5' and 3' ends blocked to ligation. Orientation of the cassette will determine which. DNA
strand will havc a free 3' end to initiate RCR. This will allow each strand to be replicated in about 50 fo of cases.
935 DDDDDDDDDXLLLLLLLLI_LLLXDDDDDDDDDDD
DDI3DDDDDCtOLLLLLI.,(..LLLLLODDDDDDDDDDD
DDDDDDDDDOLLLL[.,LLLLLLLODDDDDDDDDDD
DDDDDDDDDX.I.,I,LLLLLLLLLLXDDDDDDDDDDD

94o D-DNA., L= adaptor, X- blocked ligation site, C)= open to ligation [001131 As will be appreciated by those in the art, there are several ways to forrn circularized adaptorltarget sequence components. In one embodiment, a CircLigase rm enzyme is used to close single stranded polynucleotide circles without template.
945 Alternatively. a bridaing template that is complementary to the two termini of the linear strand is used. In some embodiments, the addition of a first adaptor to one termini of the target sequence is used to design a cotnplementary part of the bridging template. `I'he other end may be universal template DNA containing degenerate bases for binding to all genomic sequences. I-Iybridization of the two termini followed by ligation results in a circularized 950 component. Alternatively, the 3' end of the target molecule nn.ay be modified by addition of a poly-dA tail using terminal transferase. The modified target is then circularized using a bridging template complementary to the adaptor and to the oligo-dA tail.
1001141 In another ei-nbfdiment, biotin is incorporated into each template oligonucleotide used to guide ligation. This allows for easy removal of templates, for examp[e by applying 955 high temperature melting, which removes the templates without removing fonned circles.
Thcse longer oligonucleotides can serve as primers for RCR oi- be used for other purposes such as inserting additional cassettes.
1001151 In another embodiment, the target DNA may be attached to some solid support such as magnetic beads or tube/plate well walls to allow removal of all templates or adaptors that 96o are not covalently ligated to the target DNA. 'I'arget ssDNA may be attached using a support with random primers to extend and create about 20-80 bases of dsDNA. The extension length may be controlled by time or by the amount of ddNTPs. Another approach is to ligate an adaptor to one end of the ssDNA and then size select DNA with the adaptor ligated to the ssDNA, and at the same time removing free adaptor. In this case an anchor sequence about 965 10-50 bases in length complementary to part of the adaptor may be attached to the support to capture DNA and use it for subsequent steps. This anchor molecule may have additional components to increase hybrid stability, such as the incorporation of a peptide nucleic acid.
Another method for attaching singic stranded DNA is by utilizing a single stranded DNA
binding protein attached to the support.
970 1001161 In one method of circularization, illustrated in Fig. 2A, after genomic DNA (200) is fragmented and denatured (202), single stranded DNA fragments (204) are first treated with a terminal trarr4ferase (206) ;, :i`tach a poly dA tails (208) to 3-prime ends. This is then oligon.ucleotide (21 0) that is complementary to the poly dA tail at one end and 975 complementary to any sequence at the other end by virtue of a segment of degenerate raucleotides. Duplex region (214) of bridbing oligonucleotide (210) contains at least a primer binding site for RCR and, in some embodiments, sequences that provide complements to a capture oligonucleotid.e, which may, be the same or different from the primer binding site sequence, or which may overlap the primer binding site sequence. rI'he 980 length of capture oligonuclcotides may vary widely, In one aspect, capture oligonucleotides and their complements in a bridging oligonucleQtide have lengths in the razzge of from 10 to 100 nucleotides; and more preferably, in the range of from 10 to 40 nucleotides. In some embodiments, duplex region (214) may contain additional elements, such as an oligonucleotide tag, for example, for identif'ying the source nucleic acid from which its 985 associated DNA fragment came. "I'hat is, in some enibodiments, circles or adaptor ligation or concatemers from different source nucleic acids may be prepared separately during wllich a bridging adaptor containing a unique tag is used, after which they are mixed for concatenier preparation or application to a surface to produce a random array.
`I'he associated fragments may be identified on such a random array by hybridizing a labeled tag g9 complement to its corresponding tag sequences in the concatemers, or by sequencing the entire adaptor or the tag region of the adaptor. Circular products (218) may be conveniently isolated by a conventional purification column, digestion of non-circular DNA
by one or more appropriate exonucleases, or both.
1001171 DNA fragyiients of the desired sized range, e.g. 50 - 600 nucleotides, can be 995 circularized using circularizing enzymes, such as CircLigase, as single stranded DNA ligase that circularizes single stranded DNA without the need of a template. A
preferred protocol for formin; single stranded DNA circles c-ornprising a DNA fragrnent and one or more adaptors is to use a standard ligase, such as T4 ligase, for ligating an adaptor to one end of a DNA fragment followed by application of CircLigase to close the circle.
000 [001181 In an exemplary method, a DNA circle comprising an adaptor oligonucleotide and a target sequence is generated using T4 ligase utilizes a target sequence that is a synthetic olzgonucleotide TIN (sequence : 5'-NNNNNNNNGCA'I'AN C ACGANGI'CA'I NATCGTNCA.AACG"I'CA(3'I'CCANGAA`I'CN
AGATCC.ACT`I'AGANTGNCGNNNNNNNN-3'}(SI=Q ID NO: 1}. Tbe adaptor is made up 0 05 of 2 separate oligonucleotides. The adaptor olil;onucleotide that joins to t.be.. 5' end of TI N is BR2-ad (seque--Ic,: : ~'-'I`~ ~.'T~~C.~y`~fC ~C~G. kAA.~~.~(-s~.At-~~. . , '. ;AC'A'f"fAx~~~;~~.~~ ~'~
3{}

(SEQ ID NO: 2) and the adaptor oligonucleotide that joins to the 3' end of T1N
is CJR3-ext (sequence : 5'-ACCTI'CAGACCAGAT-3' ) (SEQ ID NC?: 3).
loto [001 1.91 UR3-ext contains a type Ils restriction enzyme site {Acu 1:
CT'I'CAG} to provide a way to linearize the DNA circular for insertion of a second adaptor. BR2-ad is annealed to BR2-temp (sequence 5'-NNNNN'VtiGTCCGTTAA"hG"I'CC"hCAG-3') (SEQ ID NO: 4) to form a doublc;-stranded adaptor BR2 adaptor, LTR3-ext is annealed to biotinylated tr R3-ternp (sequence 5'-[BIOTIN] ATCTGGTCTGAAGGI-NNNNNNN-3') (SEQ ID NO: 5) to [ttt -S form a double-stranded adaptor UR3 adaptor. I pmol of target TIN is ligated to 25 pmol of BR2 adaptor and 10 pznol of UR3 adaptor in a single ligation reaction containing 50mM
Tris-Cl, pl-17.8, 10% PEG, ImM ATP, 50 mg.rf, BSA, 10m.M MgCI2. 0.3 unit/ul T4 DNA
ligase (Epicentre }3iotechn.ologies, WI) and 10 mM DTT) in a final volume of 10 l. The ligation reaction is incubated in a temperature cycling program of i 5'C for 11 cnin, 37 C for 020 1 min repeated 18 times. The reaction is terminated by heating at 70 C for 10 n1in. Excess BR2 adaptors are removed by capturing the ligated products with streptavidin magnetic beads (NTew Enbland Biolabs, MA). 3.3 p] of 4x binding buffer (2M NaCI, 80 rnM
Tris I-ICI
pI-I 7.5) is added to the ligation reaction, which is then combined with 15 Ag of'streptavidin n-iagnetic beads in a 1 x binding buffer (0.5M NaCl, 20 mM `1`ris HCl pH7.5).
After a 15 025 minute incubation in room temperature, the beads are washed twice with 4 vOlLIrnes of fow salt buffer (0.15M NaCI, 20 mM Tris HCl pH 7.5). Elution buffer (10 mM Tris HCI pII 7.5) is pre-warmed to 70 deg, 10 pl of which is added to the beads at 70 C for 5 min. After magnetic separation, the supernatant is retained as primary purified sample.
Tb.is sample can be further purified by removing the excess UR3 adaptors with magnetic beads pre-bound 030 with a biotinylated oligonucleotide BR-re-bio (sequence : 5'-[BIO'I'IN]CITTTGTCTTCCTAACATCC-3') (SEQ ID NO: 6) that is reverse complementary to BR2-ad similarly as described above.
[001201 The concentration of the adaptor-target ligated product in the final purified sample can be estimated by urea polyacrylamide gel electrophoresis analysis. The circularization is 035 carried out by phosphorylating the ligation products using 0.2 unit`pl "l-4 pc3[ynucleotide kinase (Epicc;ntre Biotechnologies) in I mM ATP and standard bufTer provided by the stipplier, and circularized with ten-fold molar excess of a splint oligonucleotide t`R3-closing-88 (sequence 5'-AGATGA_I`AATCTGGTC-3') (SEQ 1D NO: 7) using 0.3 unit~'pl of T4 DNA ligase (Epicentre Biotechnologies) and 1 mM ATP. The circularized product is )40 1?erforzning RCR reactions.

1001211 In another exemplary embodiment, which is illustrated in Fig. 2A, adaptor oligonucleotides (1604), are used to form (I6[l8) a population (1608) of DNA.
circles by the jnethod illustrated in Fig, 2A. In one aspect, each member of population (1608) has an adaptor with an identical anchor probe binding site and type Ils recognition site attached to a 1045 DNA fragment from source nucleic acid (1600). 'I'be adaptor also may have other functional elements including, but not limited to, tagging sequences, sequences for attachment to a solid surface, restriction sites, f'unctionalization. sequences, and the like.
Classes of DNA
circles may be created by providing adaptors having different anchor probe binding sites.
1001221 After DNA circles (Fig. (2A) 1608) are formed, further interspersed adaptors are 1050 inserted as illustrated generally in Fig. (2A) to form circles (1612) containing interspersed adaptors. To these circles, a primer and rolling circle replication (RCR) reagents can be added to generate (1614) in a conventional RCR reaction a population (1616) of concatemers (1617) of the complements of the adaptor oligonucleotide and DNA
fragments.
This population c-an then be isolated or otherwise processed (e.g. size selected) (1618) using 1055 conventional techniques, e.g. a conventional spin column, or the like, to form population (1620) for analysis.

100123] To demonstrate that the formation of multiple -adaptor DNA circles is feasible a synthetic target DNA of 70 bases in length and a PCR derived fragment of 200-300 bp in length may be obtained. A single stranded PCR fragment can be simply derived from a 1060 dotible stranded product by phosphorylation of one of the primers and treatment with lambda exonuclease to remove the phosphorylated strand. The single stranded fragment may be ligated to an adaptor for circularization. Polymerization, type IIs restriction enzyme digestion and re-ligation with a new adaptor may be performed as described herein.
[001.24] Demonstration that the process was successful may proceed by RCR
amplification 065 of the final derived circles. Briefly, the DNA circles are incubated with primer complementary to the last introduced adaptor and phi29 polymerase for I hour at 30 C to generate a single concatemer molecule conzprising hundreds of repeated copies of tlle original DNA circle. Attachment of the RCR products to the surface of coverslips inay proceed by utilizing an adaptor sequence in the concatemer that is cornplei-nentary to an fl~o attached oligonucleotide on the surface. I-lybridization of adaptor Linique probes may be used to demonstrate that the individual adaptors were incorporated into the circle and ultimately the RCR product. "I`o demonstrate that the adaptors were incorporateci at the expected p :~. 'i~.'~ `~i~i.tl~--f i~'.1rclE c:=,L.'E",ni',0 b 11' ~ Ã:

1075 probe that recol;nizes the terminal sequence of the adaptor. Cloning and sequencing nlay also be used to verify DNA integrity.
1001251 ln one embodiment, a template used for circle formation can also be used as a primer to create localized dsDNA. The schema is simplified bv generatiiig clean ssDNA
after each circle cutting which allows the use of the sanie circle closing chemistry for each 1080 adaptor incorporations.
1001261 In one embodiment, a solution of DNA fragments with sticky ends or biurit ends is prepared for making DNA circles. The traditional method to avoid making circles with more than one DNA molecule is to perform ligation in a large volume at a low concentration of DNA lragments where intermolecular ligation is unlikely.
1085 1001271 In a preferred embodiment, the ligation reaction does not require a large volume.
`1`his embodiment involves a slow addition of aliquots of DNA fragments into a regular size ligation reaction. Fast rnixing of the DNA aliquot and the reaction minimizes multi-mer fon-nation,. The DNA fragments can be prepared in a ligation mix without ligase or in water or "1'1-~~-like buffer. Typically, the DNA volume is equal to or lower than the initial volume of 1090 ligation reaction. DNA may be in a large volume in water or simple buffer (such as TE
buffer) if the ligation reaction evaporates with the speed of adding the DNA
sample. The evaporation may be simplified by using thermo-stabile ligase.
1001281 In one embodiment, the method of circularization involves diluting a small aliquot of DNA into a regular ligation reaction (such as Ã1.1-0.5 l in 10-50 1 provides over 100 fold 095 dilution) and waiting for sufficient time to allo", a majority of the DNA
to form circles, followed by addition of a second aliquot. In another embodiment, DNA fragments are slowly and continuously added.
1001291 Various physical implementations of the process are possible, such as manual or automated pipetting at a certain frequency, the use of drippers (gravity or positive pressure), ioo piezo or acoustic spiting or nanodroppers, cavro-pumps that can deliver drops as small as 30 nl. In one embodiment 10 pmols in 100 p.I reaction having maximal temporal concentration of I fmol:`ul is processed using a consecutive addition of 100 aliquots. In another embodiment, 10 pmols are in 30-50 p1 aliquots. The time to circularize >70-80%
of DNA
fragments in one, aliquot depends on ligase concentration, type of ends (sticky 1, 2, or 4 5 bases or blunt) and to some extent temperature (movements and hybrid stability of sticky ends). In a preferred embodiment, the total Ãinie of the reaction is approximately 4-1 6 hours.
1001301 In (,nEa :.-nbc3dimunt, a iaf~ ~4 r nzyrrte -~n . solid sa.tp -_ ;,rc~
as a f ~

porous container using methods known in the art. To prevent ligation between fragments lito (rather than circularization), methods kn.ow-n in the art for temporarily blocking the DNA
may be used, including but not limited to the use of non-ligatable DNA with matching sticky ends or ssDNA end binding proteins.
1001311 To increase the efficiency of flow-through of a small reaction volume, in one embodiment the reaction volume is dispensed under non-evaporating, conditions, for 1115 example by using small drople-ts. Non-evaporating conditions can also be established by regulating humidity, temperature of the support ambient, and through design of the composition ofrea.ction buffer. In en exemplary embodiment, 10 pl drops are dispensed by piezo spitting, (-20 x 20 x 20 microns). With no spreading this is equivalent to a 20 znicron thick flow cell. Spreading can be proinoted to fiirt.ber reduce thickness of the volume to 112o about 5-10 microns. To cover one cm '` using 10 pl drops with zero spreading, 100 x 50 x 50 = 250,000 drops can be used.
[001321 In addition to piezo approach other forms of delivery of low amount of buffer per large surface can be used, such as by coiita.eting the support with a porous material filled with reaction buffer or to move a long slit across the surface with a few 10-30 micron 1125 openings allowint), dispensation of the buffer.
[00133] One exemplar_v method of circularization involves ligation of a single adaptor to dsDNA using two blocked complementary strands. In this method, two complementary strands of an adaptor are independently prepared. A rnatching blocking oligo that has uracils and can not be ligated to tar-et DNA is also made for each of the two complementary 130 strands. A dsDNA prodtÃct comprising of one adaptor strand and one blocking oligo is assea-nbled. Two assembled dsDNA constructs are desi(ined that can not ligate or hybridize one to another; the constructs may be blunt end or may have a T overhang or other overhangs for ligation to DNA targets. A mixture of these two constructs is ligated to blunt end dsDNA or DNA with corresponding sticky ends. About 50% of DNA will have one of 135 each construct; the other 50% will have two of the same construct. The blocking oligo is then degraded, and the circle is closed by hvbridization of complimentary strands and ligation.
(00134] In one embodiment, the adaptor may be palindromic to avoid distinction of orientation. Suc.h an approach can provide a better yield than A/T ligation approach, 14t) depending on blunt end ligation efficiency and concentration of DNA in A/T ligation reaction. In a i~urtber embodiment, four instead of two ssDNA adaptor components are used.

EVlethads for creating concatemers 1001351 In one aspect of the invention, single molecules comprise concatemers of polynucleoticies, usually polyrzucleotide analytes, i.e. target sequences, that have been 1145 produce in a conventional rollincy circle replication (RCR) reaction.
Guidance for selecting conditions and reagents for RCR reactions is available in many references available to tiiose of ordiuary skill, as evidence by the following that are incorporated by reference: Kool, U.S.
patent 5,425,180; Lizardi, U.S. patents 5,854,033 and 6,143.495; Landegren, U.S. patetit 5,871,921; and the like. Gerzerally, RCR reaction components comprise single stranded 1150 DNA circles, one or more primers that anneal to DNA circles, a DNA
polymerase having strand displacement activity to extend the 3' ends of primers annealed to DNA
circles, nucleoside triphosphates, and a conventional polymerase reaction buffer. Such components are combined under conditions that permit primers to anneal to DNA circles and be extended by the DNA polymerase to form concatemers of DNA circle complements. An exemplary 11.55 RCR reaction protocol is as follows: In a 50 ut: reaction mixture, the following ingredients are assembled: 2-50 pmol circular DNA, 0.5 units/ L phage (p29 DNA polymerase, 0.2 }tg/ L BSA, 3 mM dNTP, 1 X (p29 DNA polymerase reaction buffer (Amershain).
The RCR
reaction is carried out at 30"C for 12 hours. In some embodiments, the concentration of circular DNA in the polymerase reaction may be selected to be low (approximately 10-100 1160 billion circles per ml, or 10-100 circles per picoliter) to avoid entanglement and other intermolecular interactions.
10013(] Preferably, concatemers produced by RCR are approximately uniform in size;
accordingly, in some embodiments, methods of making arrays of the invention may include a step of size-selectint) concatemers. For example, in one aspect. concatemers are selected 165 that as a population have a coefficient of variation in molecular weight of less than about 30%; and in another embodiment, less than about 20%. In one aspect, size uniformity is further improved by adding low concentrations of'chain tc;rininatars, such ddN"I'i's, to the RCR reaction mixture to redLice the presence of very large concatemers, e.g.
produced by.
DNA circles that are synthesized at a higher rate by polymerases. In one cmbod.iment, 170 concentrations of ddNI`Ps are used that result in an expected concatemer size in the range of from 50-250 Kb. or in the ran.Le of from 50-100 Kb. In another aspect, concatemers may be enriched for a particular size range using a conventional separation techniques, e.g. size-exclusion chromato2raphy, membrane filtration, or the like.

1001371 An exemplary method for producing concatemers is illustrated in Fig.
2A. After 1175 DNA circles (1608) are formed, furtber interspersed adaptors are inserted as illustrated generally in Fig. (2A) to form circles (16 12) containing interspersed adaptors. "l-o these circles, a primer and rolling circle replication (RCR) reagents can be added to generate (1614) in a conventional RCR reaction a population (1616) of concateme-rs t 1617) of the complements of the adaptor oligonucleotide and DNA fragments. Tb.is population can then 1180 be isolated or othenvise processed (e.g. size selected) (1618) using conw-entional techniques, e.g. a conventional spin column, and the like, to form population (1620) for analysis.
1001381 Target polynLicleotides may be generated from a source nucleic acid, such as genomic DNA, by fragmentation to produce fragments 0.2-2 kb in size, or more preferably, 03-0.6 kb in size, which then may be circularized for an RCR reaction.
1185 (00139] ln another aspect, the invention provides methods and compositions for generating concaterners of a plurality of target polynucleotides containing interspersed adaptors. In one embodizn.ent, such concatemers may be generated by RCR, as illustrated in Figs. 1C-lI).
1001401 Rolling circle replication is a pref'erred method of creating concatemers of the invention. The RCR process has been shown to generate multiple continuous copies of the t9o M13 genorne. (Blanco, et al., (1989) JBiol C'hc.jrn 264:8935-8940). In this system, the desired DNA fragment is `'cloned" iaito a DNA adaptor and replicated by linear concatemerization. The target DNA is immediately in a form suitable for hybridization and enzymatic methodologies without the need to passage through bacteria.
1001411 The RCR process relies upon the desired target molecule first being formed into a 195 circular substrate. This linear amplification uses the original DNA
molecule, not copies of a copy, thus ensuring fidelity of sequence. As a circular entity, the molecule acts as an endless template for a strand displacinc, polymerase that extends a primer complementary to a portion of the circle. The continuous strand extension creates lona, single-stranded DNA
consistim, of hundreds of concatemers comprising multiple copies of sequences 200 complementary to the circle.
rVethods or creatin urnm 100I421 In one embodiment, emulsion PCR is used to generate amplicoiis for disposal onto an array. As illustrated in 1"ig. (1B) after breaking emulsion (1505), beads containing clones of the adaptored sequences may be arrayed (1520) on a solid surface (1 -522) for sequence 205 analysis. Such array of beads may be random, as illustrated in Fig. 1 F.
where the locations are n arr< ace predetermined pattern of binding sites (1524), even though the distribution of beads on such sites is randomly determ.ined. Both of such distributions are referred to herein as "random arrays."

1210 1001431 7,o achieve compact, dense bundles of the DNA in the form of sub-tmicron spots, a region of the amplified molecule for hybridization to a capture probe attached to the glass surface can be utilized, 14undreds of capture probe molecules (spaced about 10 n.zn apart) can keep hundreds of concatenated copies of a target molecule tightly bound to a glass surface area of less than 500 nm in diaineter. In one embodiment, glass activation chemistry is 1215 applied that creates a monolayer of isothiocyanate reactive groups for attaching amine modified capture oligonucleotides.

1001441 Generally, densities of single molecules are selected that permit at least twenty percent, or at least thirty percent, or at least forty percent, or at least a majority of the molecules to be resolved individually by the signal generation and detection systems used.
1220 In one aspect, a density is selected that permits at least seventy percent of t13e single molecules to be individually resolved. In one aspect, whenever scanning electron microscopy is emptoyed, for example, with molecule-speciiic probes having gold nanoparticle labels, e.g. Nie et al (2006), Anal. Chem., 78: 1528-1534, which is incorporated by reference, a density is selected such that at least a majority of single molccules have a 225 nearest neighbor distance of 50 nm or greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 100 nm or greater. In another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a density is selected such that at least a majority of single molecules have a nearest neighbor distance of 200 nm or 230 greater; and in another aspect, such density is selected to ensure that at least seventy percent of single molecules have a nearest neighbor distance of 200 nm or greater. In still another aspect, whenever optical microscopy is employed, for example with molecule-specific probes having fluorescent labels, a densitv is selected such that at least a majority of sinole molecules have a nearest neigghbor distance of 300 nm or greater; and in another aspcct, such 235 density is selected to cnsure that at least seventv percent of single molecules have a nearest neighbor distance of 300 nm or greater, or 400 nm or greater, or 500 nm or (ireater, or 600 nm or greater, or 700 nm or greater, or 800 nm or greater. In still another embodiment, whenever optical inicroscopy is used, a density is selected sucEi that at least a majority of , sZnele 'r"ler.,_Oos he. - t -t i:rq=JI"cr di ; `aa_,~ _ 1 -= r.,_ _ s nnce of at fvaee ~_._ . ~ . ~: _ ....
'40 'pc.t ,7 are disposed on a surface so that the density of separately detectable polymer nio[ecules is at least 1000 per m`, or at least I fl,000 per ~m". or at [east 100,[?00 per l.tm`.
1001451 In another aspect of the invention, the requirement of selecting densities of randomly disposed single molecules to enstEre desired nearest neighbor distances is obviated 1245 by proviÃiizig on a surface discrete spaced apart regions that are substantially the sole sites for attacb.inw~ single molecules. That is, in such embodiinerats the regions on the surface between the discrete spaced apart regions, referred to herein as "inter-regional areas," are inert in the sense that concatemers, or other macromolecular structures, do not bind to such regions. In some embodiments, such inter-regional areas may be treated with blocking 1250 agents, e.g. DNAs unrelated to concatemer DNA, other polymers, and the like. Gen.erally, the area of discrete spaced apart regions is selected, along with atiachment chemistries, macromolecular structures employed, and the like, to correspond to the size of single molecules of the invention so that when single molecules are applied to surface substantially every region is occupied by no more than one single molecule. The likelihood of having 255 only one single molecule per discrete spaced apart reoion may be increased by selecting a density of reactive functionalities or capture oligonucleotides that results in fewer such moieties than their respective complements on single molecules. Thus, a single molecule will "occupy" all linkages to the surface at a particular discrete spaced apart region, thereby reducing the chance that a second single molecule will also bind to the same region. In 260 partieular, in one embodiment, substantially all the capture oligonucleotides in a discrete spaced apart region hybridize to adaptor oligonucleotides a single macromolecular structure.
In one aspect, a discrete spaced apart region contains a number of reactive functionalities or capture oligonucleotides that is from about ten percent to about fifty percent of the number of complementary functionalities or adaptor oligonucleotides of a single molecule. The 265 length and sequence(s) of capture oligonucleotides may vary widely, and may be selected in accordance with well known principles, e.g. Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 227-2-59 (1991); Britten and Davidson, chapter 1 in Hames et al, editors, Nucleic Acid Hybridization: A Practical Approach (IRL Press, Oxford, 1985). In one aspect, the lengths of capturÃ,~ oligonucleotides are in a range of from 6 to 30 nuclÃ:otides, 220 and in another aspect. within a range of from 8 to 30 tiucleotides. or from 10 to 24 nucleotides. Lengths and sequences of capture oligonucleotides are selected (i) to provide effective binding of maeromolecular structures to a surface, so that losses of macromolecular structures are ;, _~ dt c ratior~s, sticl~ as and 4iij 3$

to avoid interference with analytical operations on analyte inolecules, particularly when 275 analyte molecules are DNA fragments in a concatemer. In regard to (i), in one aspect, sequences and lengths are selected to provide duplexes between capture oligonucleotides and their complements that are sufficientlv stable so that they do not dissociate in a stringent wash. In regard to (ii j, if DNA fragments are frorn a particular species of organism, then databases, when available, may be used to screen potential capture sequences that may form 280 spurious or undesired hybrids with DNA fragments. Other factors in selecting sequences for capture oligonucleotides are similar to those considered in selectint, primers, hybridization probes, oligonucleotide tags, and the like, for which there is ample guidance, as evidenced by the references cited below in the Definitions section.
[001461 In one aspect, the area of discrete spaced apart regions is Iess than I I.Im'; and in 285 another aspect, the area of discrete spaced apart regions is in the range of from 0.04 q.rri'` to 1 m 2 ; and in still another aspect, the area of discrete spaced apart regions is in the range of from 0.2 Pm' to 1~Im'. In another aspect, when discrete spaced apart regions are approximately circular or square in shape so that their sizes can be indicated by a single linear dimension, the size of such regions are in the range of from 125 nm to 250 nm, or in 290 the rancTe of frorn 200 nm to 500 nm. In one aspect, center-to-center distances of nearest neighbors of such regions are in the range of from 0.25 m to 20 m; and in another aspect, such distances are in the range of f-rotn 1~trn to 10 ~irn, or in the ran-e from 50 to 1000 nrn.
Preferably, spaced apart regions for immobilizing concatemers are arranged in a rectilinear or hexagonal pattern.
295 1001471 In one embodiment, spacer DNBs are used to prepare a surface for attachment of test DNBs. The surface is first covered by the capture oligonucleotide complementary to the binding site present on two types of synthetic DNBs; one is a capture DNB, the other is a spacer DNB. The spacer DNBs do not have DNA segments complementary to the adaptor used in preparation of test DNBs and they are used in about 5-50, preferably l Ox excess to 300 capture DNBs. The surface with capture oligonucleotide is "saturated" with a rnix of synthetic DNBs (prepared by chain ligation or by RCR:) in which the spacer DNBs are used in abotit 10 -fold (or 5 to 50-fold) excess to capture DNBs. Because of the -10: I ratio between spacer and capture L3NBs, the capture DNBs are mostly individual islands in a sea of spacer DNBs, 'I'he 10:1 ratio provides that two capture DNBs are on average separated by two spaccr DNBs. If DNBs are, about 200 nm in diameter, then two capture DNBs are at molecular structures that have a binding site complementary to a region of the capture DNBs but not present on the spacer DNBs.
1001481 Capture DNBs may be prepared to have fewer copies than the number of binding 1310 sites in test DNBs to assure, single test DNB attachment per capture DNB
spot. Because the test DNA can bind only to capture DNBs, an array of test DNBs may be prepared that hav-e high site occrtpancy without congregation. Due to random attachment, some areas on the surface may not hazfe any DNBs attached, but these areas with free capture oligonucleotide may not be able to bind test DNBs since they are designed not to have binding sites for the 1315 eaptLire oligonucleotide. Arrays of the invention may or may not be arranged in a grid pattern.
[001491 ln one aspect, a high density array of capture o[igonucleotide spots of sub micron size is prepared using a printing head or imprint-master prepared from a bundle, or bundle of bundles, of about 10,000 to 100 million optical fibers with a core and cladding material. By 1320 proper pulling and fusing fibers, a unique material may be produced that has about 50-1000 nm cores separated by a similar or 2-5 fold smaller or larger size cladding material. In one ernbodiment, differential etching (dissolving) of cladding material provides a nano-printing liead having a very large number of nano-sized posts. This printing head may be used for depositing oligonueleotides or other biological (proteins, oligopeptides, DNA, aptamers) or 325 chemical compounds such as silane with various active groups.
[00150] In one embodiment the glass fiber tool may be used as a patterned support to deposit oligonucleotides or other biological or chemical compounds. In this case only posts created by etching may be contacted witli material to be deposited. In another embodiment, a flat cut of the fused fiber brlndle may be used to guide light through cores and allow light-330 induced chemistry to occur only at the tip srirface of the cores, thus eliminating the need for etching. In both ernbodiments, the same support may then be used as a light guiding/collection device for imaging fluorescence labels used to tag oligonucleotides or other reactants. This device provides a large field of view with a large numerical aperture (potentially > 1 ).
335 1001511 Stamping or printing tools that perform active material or oligonucIeotide deposition may be used to print 2 to 100 different oligonucleotides in an interleaved pattern.
This type of oligonucleotide array may be used for attaching 21 to 100 different DNA
populations, such as populations derived from different source DNA. They also may be used lor par...11c' frotn sub-iiah" .:scguy:rn b; DNA spÃ,. .. _ ,, r~ =s, ... i, .... . i~

I;. :c 'f >sed b ~ . Ã

and read 2 bases by a combination of 5-6 colors and using 161igation cycles or one ligation cycle and 16 decoding cycles.
(001.52] In embodiments of the invention, photolithography, electron beam lithography, nano imprint lithography, and nano printing may be used to generate such patterns on a wide 1345 variety of surfaces, e.g. Pirrung et a1,U.S. patent 5,143,854; Fodor et al, U.S. patent 5,774,305; Guo, {2404} Journal of Physics D: Applied 1'hysics, 37: R I23-14I
;which are incorporated herein by reference. These techniques can be used to generate pattern.s of features on the order of 11`10''' of a micron and have been developed for use in the semiconductor industry. In a preferred embodiment, a single "masking"
operation is 1350 performed on the DNA array substrate, as opposed to the 20 to 30 masking operations typically needed to create even a simple semiconductor. Using a single masking operation eliminates the need for the accurate alignment of many masks to the same substrate. There is also no need for doping of materials. Minor defects in the pattern may have little to no effect on the usability of the array, thus allowing production yields to approach 100%.

I355 1001.531 In one embodiment, high density structured random DNA array chips have capture oli-orzucleotides concentrated in small. sefrefated capture cells aligned into a rectangular grid formation (Fig. 4). Preferably, each capture cell or binding site is surrounded by an inert surface and may have a sufficient but limited number of capture molecules (100-400). Each capture molecule may bind one copy of the rnatcbing adaptor sequence on the RCR
360 produced DNA concatemer. Since each concatemer contains over 1000 copies of the adaptor sequence, it is able to quickly saturate the binding site upon contact and prevent other concatemers from binding, resulting in exclusive attachment of one RCR product per binding site or spot. By providing enough RCR products almost every spot on the array may contain one and only one unique DNA target.
365 [001541 RCR "niolecular cloning" allows the application of the saturation/exclasion (single occupancy) principle in making random arrays. The exclusion process is not feasible in making single molecule arrays if an in situ amplification is alternatively applied. RCR.
coricatemers provide an optimal size to form sanaIl non-mixed DNA spots. Each concatemer of about 100 kb is expected to occupy a space of about 0.1 x 0.1 x 0.1 ~Im, thus allowing 370 RCR products to fit into 100 nm capture cells. One advantage of RCR
products is that the single stranded DNA is ready for hybridization and is very flexible for forming a randomly coiled ball of DNA. The 1000 copies of DNA target produced by RCR provide much higher ~,. :r tbar~. is possible with arial 1001551 `l'here are methods known in the art for generating a patterned DNA
chip. In a 1375 preferable cmbodiment, all spots on the chip have the same capture oligonacleotides and a 0.2-0.3 micron spot size at 0.5 micron pitch. Nano-printing approaches may be used for producing such patterrls, as they do not require development of new oligonucleotide attachment chernistrv.
[00156] Nano-imprint technologies rely on classic photolithographic techniques to produce [3Sa a master mold. The master mold is then replicated using polymers such as PMMA or PBMS. These polymers, upon curing, form a nepative mold oftbe master. The mold is then used to "print" patterns of material on a substrate. Tbe nano-imprint technique can be used to create protein features on glass, silicon, and gold surfaces. In an exemplary embodiment, a master mold is tised to generate many stamping devices and each stamping device can 385 generate many prints of chemicals (such as oligonucleotide solution, oligonucleotide binding or glass activation chemicals). Advanced nano-printing techniques can produce features as small as 10 nrn, thus, features appropriate for fluorescent detection that are >200 nm in sire, including features 300-500 nm at 1000 microns center to center, can be produced routine[y.
1001571 Various chemical modifications can be used to alter surface properties, increasing 390 the compatibility of the master mold with a wide range of materials, thus allowing the use of a small feature, low-density mold to create high density arrays. In one embodiment, a mold with a 4um feature pitch can. be used to create a one um feature pitch on the substrate by printing the same substrate 16 times in a 4 by 4 grid.
(00158] In one aspect, a method of creating DNA arrays involves the use of a thin laver of 395 photo-resist to protect portions of the substrate surface during a functionalization process.
The patterned photo-resist is removed after functionalization, leaving an array of activated areas. The second approach involves attaching a monolayer of modif-ied oligonucleotides to the substrate. "I'he oligonucleotides are modified with a photo-cleavable protecting group.
These protecting grotips can be removed by exposure to an illumination source, allowing 400 patterned ligation of a capture oli(lonueleotide for attachment of DN13s by hybridization.
[00159] In another embodiment, a commercially available, optically flat, quartz wafer is spin coated with a 100-500 nm thick layer of photo-resist. The photo-resist is baked on to the quartz wafer, and an image of a reticle with a pattern of spots to be activated is projected onto the surface of the photo-resist, nsing a machine commonly called a stepper. After 4 4s exposure, the photo-resist is developed, removing the areas of the projected pattern which were exposed to the 1.;V sourcc, This is accotiiplished by plasma etching, a dry developing technique capable of producing very fine detail. "l"he wafer is then baked tc) strengthen the remaining photo-resist.
[00160] After baking, the quartz wafer is ready for functionalization. `I'he wafer is then 4W subjected to vapor-deposition of 3-arninopropyldirneihylethoxysilane, the same monomer used in the current functionalization process. `I'he densitv of the amino functionalized monomer can be tightly controlled by varying the concentration of the monomer and the time of exposure of the substrate. Only areas of quartz exposed by the plasma etching process may react with and capture the monomer. The wafer is then baked aiain to cure the 415 monolayer of amino-functionalized monomer to the exposed quartz. After baking, the remaining photo-resist may be removed using aeetone. Because of the difference in attachment chemistry between the resist and silane, aminosilane-functionalized areas on the substrate may remain intact through the acetone rinse. These areas can be further functionalized by reacting them with p-phenylenediisothiocyanate in a solution of pyridine 420 and N-N-DiMethlyFormamide. The substrate niay then be compatible with amine-modified oligonucleotides. Altematively, oligonucleotides can be prepared with a 5'-carboxy-m.odifier-c10 (Glen Research: http://w-ww.glenres.com%l'roductFiles/I0-I935.html). `I'his technique allows the oligonucleotide to be attached directly to the amine i:nfldi#ied sl-pport, thereby avoiding additional functionalization steps.
425 1001611 In another embodiment, a nano-imprint lithography (NIL) process is used which starts with the production of a master imprint tool. This tool is produced using high-resolution e-beam lithography, and can be used to create a large number of imprints, depending on the NIL polymer utilized. For DNA array production, the quartz substrate would be spin coated with a layer of resist, this layer commonly called the transfer layer. A
430 second type of resist is then applied over the transfer layer, this layer is commonly called the imprint layer. The master imprint tool theii makes an impression oD the imprint layer. The overall thickness of the imprint layer is then reduced by plasma etchin- until the low area's of the imprint reach the transfer layer. Because the transfer layer is harder to remove than t.be imprint layer, it remains largely untouched. The imprint and transfer layers are then 435 hardened by heating. 'The substrate is then put back into the plasma etcher tintil the low areas of the imprint reach the quartz. "I'he substrate is then derivatized by vapor deposition as described in method I a..
[001621 In another embodiment. a nano-printing method is used. Such a process uses photo.
a e the 4=t0 .Ae master mold is created as a negative image of the features required on the print head. The print heads are usually made of a soft, flexible polymer such as polydimethylsiloxane (PDMS). This material, or layers of materials baving different properties, are spin coated onto a quartz substrate. The mold is then used to emboss the features (into the top layer of 1445 resist material tinder controlled temperature and pressure conditions.
The print head is then subjected to a plasma based etching process to iznprove the aspect ratio of the print head, and eliminate distortion of the print head due to relaxation over time of the embossed material.
The print head is used to deposit a pattern of amine modified oligonucleotides onto a homogenously derivatized surface. These oligo-nucleotides serve as capture probes for the 1450 DNB's. One advantage to n.ano-printing is the abiiitv to print interleaved patterns of different capture probes onto the random array support. This can be accomplished by successive printing with multiple print heads, each head having a differing pattern, and all patterns fitting together to form the final structured support pattern. Such methods allow for positional encoding of DNA elements within the random array. For example, cojitrol DNBs 1455 containing a specific anchor sequence can be bound at regular intez-vals throughout a random array.
[00163] Electron beam lithography can also be used to create the substrate.
This process is very similar to photolithography, except the pattem is drawn directly on a special resist material using an electron beam gun. The benefit of this process is that the feature size can 460 be much smaller and more precise than with UV photolithographic methods. A
potential drawback is the amount of time required to create the pattern is on the order of hours per substrate, as opposed to a couple of seconds using photolithographic methods or less than a minute for NIL.
fU01641 In one embodiment, the arrays are produced using photo-cleavable inodifiers, also 465 referred to as protecting groups. In such a method, capture cells can be created by using commercially available photo-cleavable modifiers to oligonucleotides, such as the PC Linker :Ph.osphoramidite, available from Glen Resea.rch.. An oligonucleotide with a 5 prime photo-clea-vable protection group, in this case DiL1TO, is attached to a fu.l1v functionalized piece of quartz at the 3' terrninus. 'l'he exposed areas Iose their protecting group, leaving a 5' 470 phosphate. Using oligoiiacleotide Iigation, a capture oligonucleotide complementary to the adaptor region of RCR products is ligated to exposed phosphate oups if a teniplate oligonucleotide is provided as depicted below:
,n t'1:. 4urf~!C`` i l...pil -; ~),AI:t:ic't~;) titagg.XCO'~tgg ECc'3,p ltlr>b' Ol1g a,t 1475 gaatgaeacg...... cetgatggca (single template oIigonucleotide> ) 1001651 After ligation of the capture oligonucleotide to the deprotected surface otigonucleotides, the entire substrate can be exposed to a UV source to reznove the remaining protecting groups. "I`he free phosphate groups may be blocked by ligating hairpin I480 like oligonucleotides to prevent ligation oflabeled probes used in the sequencing process to the support oligonucleotide.
1001661 Tbe photo-resist material used in fabrication methods is generally quite hydrophobic, and the patterns made in that material consist of very small hoies. It is possible that the exposed surface of the quartz may iiot come into contact with aqueous 1485 solutions of the aniino functionalizcd monomer due to the hydrophobic effect of the pboto-resist. To avoid this problem, one embodiment of the invention is to use ultrasound to force the liquid past the small openings in the mask. It is also possible to put a small amount of surfactant, acetone, or other additive to the solution to break the surface tension of the water.
The use of solvents in this manner might swell the mask material slightly, but it would not 494 dissolve it. In the event that the resist material is incompatible with the amino-functionalized surface during the resist removal process, for instance it might react with and destroy the amine, it is possible to perform a mechanical peel of the resist material using a strong acrylic based 4dhesive on a polymer sheet.
1001671 Aiter each batch of DNA array substrates is made, it may be important to determine 495 if the batch is up to specification. Specifications may be detertnined during the mask design and biochemistry optimization phase. Quality control of each batch of sLibstrates can be performed by attaching FITC or a amine-modified oligonucleotide with any fluorescent Iabel to the reactive surface and observing the intensity and pattern of the fluorescence on the substrate surface. The overall intensity of the active regions may be proportional to the 500 density of reactive sites in the capture cells. The current microscopy system has a I o0x, 1.4NA lens that has a theoretical resolving power of about 180 nm. The sensitivity of the current image acquisition system is about 3 dye molecules per pixel, with each pixel imaging a 60x60 nm area of the substrate. It is expected to be able to attach between 10-50 capture oligonucleotides per 60 nrrm square area. This allows directly measuring, with high accuracy, 505 the attachment efficiency and grid properties of the substrate. Each capture cell may be imal;cd bv roughly 10 pixels.
10016I31 t'~in~,~ the QC data, it is possible to determine which substrate preparation steps ~t. be, Is, at this point i~the. 1,1,K , would point to uneven reaction conditions during the fiznct.ionalization process or n.on-t 51 o uniform development of the photo-resist layer. If there is bridging between cells, it would suggest that the photo-resist material delaminated from the surface of the quartz, or that somethin- went wrong during the exposure process. Problems with si"nal intensity would point to poor control of the fun.ctionalization step. Additional metrics may necessarily be developed as the process matures.

1515 Replica arrays 100169] In one aspect of the invcntion, complementary polynucleotides synthesized on a master array are transferred to a replica array. To achieve such a transfer, two surfaces may be contacted in the presence of heatincl, to denature dsDNA and free newly made DNA
strands. In another embodiment, the transfer is achieved by applying an electric field to 520 discriminatively transfer only the replicated DNA that has about 5-50 times more charge than primers. In a further embodiment, after hybridizing the transferred strand a reverse field is combined with a reduction in temperature to move primers back to the master array.
In an embodiment in which the transfer is achieved by applying an electric field, porous glass is preferably used to allow the application of the electric field.
525 1001701 In one embodiment, a capture oligonucleotide is designed to correspond to the end of an ainplicon opposite to the priming site to assure exclusive retention of the full length copies. Having a patter.n of nine or more different capture oligonuc(eotides minirnizes the chance of "cross talk" durinu DNA transfer from the master array. In one embodiment, the transfer is achieved without further amplification of DNA on the replica array; multiple 530 transfers to the same replica may also be used to generate a stronger signal. In another embodiment, multiple replicas may be generated by partial transfer frorn the master array, with DNA amplification performed in each replica array.
(001711 In an exemplary enibodiment, the substrate for the replica array contains primers for initiating DNA synthesis using template DNA attached on the first array.
After 535 contacting surfaces of the master array and support of the "to be formed"
replica array in the presence of DNA polymerase, dNTPs and suitable buffer at optimum temperature, primer molecules hybridize to the template DNA on the master array and become extended by the polymerase. A stopping agent such as dsDNA may be used to stop DNA at the end of one copy. By increasirig temperature, or by using other DNA denaturing agents, DNA
strands 540 anav separate and the replica array can be separated form the first array.
To prevent removal of oriLnnal DNA from the master array, the original DNA may be directly (or indirectly via capture olit;onucleotide) covalently attached to the master array support.

1001721 Any iiicomplete DNA that is attached to the replica array may be specifically removed after completion of the replication reaction using various methods known in the art, [545 such as through protective ligation of the completed molecules that have specific ends - the incomplete molecules can then be removed witliout losing the completed molecules.
1001731 ln one embodiment, primers cover the entire substrate surface for array preparation.
A primer density of 10,000 per micron square provides a local concentration in one rra.icron, between two supports, of similar or about 10 times higher concentration than used in PCR.
550 Primers may have very long attachment linkers to be able to reach to the DNA template on the first array's support. ln this process there is no possibility for DNA
diffusion and replica DNA spots may be only slightly larger than original spots. A very flat surface may be used to assure close proximity of two surfaces. In one embodiment, DNBs provide enough DNA
loops of about 300-500 nm and when combined with 100 nm primer linkers, allow-nty 555 tolerance of surface imperfections.
1001741 Replica arrays inay be used to produce additional replicas. Second generation replicas would have the same DNA strand as the original array.
1001751 Replica arrays inay be used for parallel analysis of the same set of DNA. fragments such as hybridization with a large iiumber of probes or probe pools. In another embodiment, 560 self-assembled DNA master chips containing genomic fragments may be replicated to generate many detection arrays that do not need to be decoded because they match the same master chip that was already decoded. Thus, replication of arrays allows us preparation of self-assembled DNA arrays with minimal decoding costs, because one master and its replicas may be used to produce thousands of final arrays.

Structure n cu ture oli os 1001761 In one embodiment, surface (Fig 1 C&.. D -- 1622) niay have attached capture oligonucleotides that form complexes, e.g. double stranded duplexes, with a segment of an adaptor oligonucleotide in the concatemers, such as an anchor bindinf, site or other elements.
5i0 In other embodiments, capture oligonucleotides may comprise oligonucleotide clamps, or like structures, that form triplexes with adaptor oli~on~.Ã:leotides, e.g. Gr~-~aznov et al, U.S.
paters.t 5,473,060. In another embodiment, surface (1622) may have reactive fuuctionalities that react with complerrsentarv functionalities on the concateruers to lorzu a covalent linkage, ~.~. t ~~tbe sa~e _ ~s Used . i cI)NAs to ~:.g _`~~ , t a:l47 1575 (2004), Genes, Chromosomes & Cancer, 40: 72-77; Beaucage (2001), Current Medicinal Chernistry, 8: 121.3-1244, which are incorporated herein by reference.
1001771 In one aspect. when enzymatic processing is not required, capture oli"onucleotides may comprise non-natural nucleosidic units and.'or linkages that confer favorable properties, such as increased duplex stability; such compounds inclucle, but ziot limited to, peptide 158o nLicleic acids (PNAs), locked nucleic acids (LNA). oligonucleotide N3'--->P5' phosphoramidates, oli.-o-2'-O-alkylribpnucleotides, and the like.

Structure of random arrrrys 1001781 In one aspect, concatemers (1620 - Fig. 1 C & D) may be fixed to surface (1622) by any of'a variety of techniques, including covalent attachment and non-covalent attachment.
1585 In one embodiment, surface (1622) may have attached capture oligonucleotides that form complexes, e.g. double stranded duplexes, with a segment of an adaptor oligonucleotide in the concatemers, such as an anchor binding site or other elements. In other cmboclirDents, capture oligonucleotides may comprise oligonucleotide clamps, or like structures, that form triplexes with adaptor oligonucteotides, e.g. Gryaznov et al. U.S. patent 5,473,0611. In I590 another embodiment, surface (1622) may have reactive functionalities that react with complementary functionalities on the concatemers to form a covalent linkage, e.g. by way of the same techniques used to attach cDNAs to microarrays, e.g. Smirnov et al (2004), Genes, Chromosomes & Cancer, 40: 72-77; Beaucage (2001), Current Medicinal Chemistry, 8:
1213-1.244, which are incorporated herein by reFerence. Long DNA molecules, e.g. several 595 hundred nucleotides or larger, may also be efficiently attached to hydrophobic surfaces, such as a clean glass surface that has a low concentration of various reactive functionalities, such as -OI1 groups.
(00179] ln one ernbodiment, complete genome sequencing uses an array comprising a 50 to 200x genome coverage of the analyzed polynucleotide fragments. For example 6 billion 600 DNBs with an average fragment length of 100 bases would contain 600 billion bases representing 1(?Ox genome coverage. In one embodiment, the array comprises 6 billion DNBs composed of 30{1-600 base long DNA fragments. The DNBs may be bound to the array substrate in a square pack arrangement at a pitch of one micron and the array substrate may be split across 16 segments. In a further cmbodimeiit, each segment contains 24 unit 605 sub arrays with each unit sub array containing 16 million bound DNBs over a 2x2 square millimeter area.

[40180] A sequencing assay which Lises 8 segments and DNB's 250 bases long may require 350 probe pools for sequencing. Various tradeofTs between fra-ment lengtb. DNB
count, pool sets, and overlap can be made to optimize sequence clualitv versus imaging time. For 1610 example, the same random array segmented into 16 segments may require 225 probe pools for sequencing. This would require fewer probe pool cycles, reducing imaging time, Additionally. DNBs can be composed of 500 base lon- fragments, requiring 3 billion DNB's to be assayed against 350 probe pools using 16 segments tested in 16 reaction chambers.
This format would produce a random array with 256x genome coverage, thus reducing the 1615 unit array size to two square m,illimeters. In one embodiment, each probe pool is corn binatorially labeled using 2 of 6 fluorophores producing up to 21 possible fluorescent label combinations. This labeling schema allows assaying against many probes simultaneously, reducing hybridization time by an order of magnitude.
[00181.1 A wide variety of supports may be used for arrays of the invention.
In one aspect, 1620 supports are rigid solids that have a surface, preferably a substantially planar sttrface so that single molecules to be interrogated are in the same plane. The latter feature permits efficient signal collection by detection optics.
1001821 In another aspect, solid supports of the invention are nonporous, particularlv when random arrays of single molecules are analyzed by hybridization reactions requiring small 1625 volumes. Suitable solid support materials include materials such as glass, polyacrylamide-coated olass, ceramics, silica, silicon, quartz, various plastics, and the like.
[00183[ In one aspect, the area of a planar surface may be in the range of from 0.5 to 4 cm'.
In one aspect, the solid support is glass or quartz , such as a microscope slide, havin- a surface that is uniformly silanized. This may be accomplished using conventional protocols, 630 e.g. acid treatment followed by immersion in a solution of 3-glycidoxypropyl trimethoxysilane, N,N-diisopropyletbylamine, and anhydrous xylene (8:1:24 v/v) at 80`C, wl-iich forms an epoxysilanized surface. e.g. Beattie et a (1995), Molecular Biotechnology, 4:
213. Such a surface is readily treated to permit end-attachment of~capture oligonacleotides, e.o. bv providing capture oligoiiucleotides with a 3' or 5' triethylene glycol pbosphorvl 635 spacer prior to application to the surface. Manv other protocols may be used for adding reactive ftinctionalities to glass and other sLirfaces, as evidenced by the disclosure in Beaucage (cited above).

1001841 Arrays of DNA taruets with interspersed adaptor(s) are not limited to single rr= a-: < <': r :,oi-tcaterraers. and arravs , i DNA of 640 partis, c ac l~ comprising t e.d in emulsion-PCR). Furthermore, methods as described herein which utilize multiple anchors or primers that can be differentially removed or otherwise discriminated are not iirraited to interspersed adaptors, i.e. they can be accomplished on samples with two "standard'", i.e.
end-ligated adaptors having a total ot'4 anchor sites.

1645 Strrtcture Qfprabe.r 1001851 The term "probes" is used in a broad sense of oligonucleotides used in direct hybridization, or as in ligation of two probes, or as in probe with an anchor, or as in a probe with an anchor probe. Probes may have only a few specific bases and many degenerate bases: for example BNNNNNNN or BBNNNNNN or NhIBBNNNN. Anchor probes may 1650 be designed as U5-1DB1-4 to read 1-4 bases adjacent to an adaptor sequence complementary to an anch or U 5 - 10 sequence.
[0(11861 The oligonticleotide probes of the invention can be labeled in a variety of ways, including the direct or indirect attachment of radioactive moieties, fluorescent moieties, colorimetric moieties, chemiluminescent moieties, and the like. Many comprehensive 1655 reviews of inethodolociies for labeling DNA and constructing I3NA
adaptors provide guidance applicable to constructing oligonucleotide probes of the present invention. SLich reviews include Kricka, Ann. Clin. Biocbem., 39: 114-129 (2002); Schaferling et al, Anal.
Bioanal. Chem., (April 12, 2006); Matthews et al, Anal. Biochem., Vol 169, pgs. 1-25 (1988); Haugland, Handbook of Fluorescent Probes and Research Chemicals, Tenth Edition 660 (Invitrogen/Molecular Probes, lnc., Eugene, 2006); Keller and fVlanak, DNA
Probes, 2nd Edition (Stockton Press, New York, 1993); and Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26: 227-259 (1991); flerrtaanson, Bioconjugate Techniques (Academic Press, New York, 1996); and the like. Many rnore particular 665 methodologies applicable to the invention are disclosed in the following sample of references: Punc, et al, U.S. patent 4,757,141; Hobbs, Jr., et al U.S. patent 5,151,507:
Cruickshank, U.S. patent 5,091,519: (synthesis of'functionalized oligonucleotidc,s for attachment of reporter groups); Jabionslci et al, N'ncleic Acids Research, 14:

(I986)(enzyme-oli-onucleotide conjugates); Ju et al, Nature, Medicint., 2.:
246-249 (1996);

670 Bawendi et aI, tI.S. patent 6,326,144 (derivatized fluorescent nanocrystals); Bruchez et al, U.S. patent 6,274,323 (derivatiz.ed fluorescent nanc>crystals); and tlle like.
[001871 In one aspec:, one or more fluorescent dyes are used as labels for the al, l;S. patent 5..188.934 (=1. ' diclzlorofluorscein dyes); Begot et al, U.S. patent 5,366,860 (spectrally resolvable rhodamine 1675 dyes); Lee et al, U.S. patent 5, 847,162 (4,7-dichlororhoda.mine dyes);
Khanna et al, U.S.
patent 4,318,846 (ether-substituted fluorescein dyes); Lee et al. U.S. patent 5,800,996 {enert-fy transfer dyes}; Lee et al.. U.S. patent 5,066,580 (xanthene dyes):
Mathies et al, U.S.
patent 5,688,648 (energy transfer dyes); and the like. Labeling can also be carried out with quantum dots, as disclosed in the following patents and patent publications, incorporated 1680 herein by reference: 6,322,901; 6,576,291; 6,423,551; 6,?51,303;

6,319,4'~16; 6.426.513;
6,444,143; 5,990,479; 6,207,392; 2002/0045045; 2003,'00 17264: and the like.
As used herein, the term "fluorescent sional generating moiety" means a si9naling means which conveys information through the fluorescent absorption andior emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence 1685 life time, emission spectrum characteristics, energy transfer, and the like.
[00188] Commercially available fluorescent nucleotide ar-ialogues readily incorPorated into the labelinc, oligonucleotides include, for exatnple, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dU'I'P (Amersham Biosciences, Piscataway, New Jersey, USA), fluorescein-I2-dL.i'I'P, tetram ethy[rhodamine-6-dUTP, Texas Redk-5-dUTP, Cascade BlueR-7-dU"l,P, BODIPY9, 6~o FL-14-dUTP, B0DIPY*R-14-dUTP, BDDIPYt TR-I4-dUTP, Rhodamine GreenT-~15-dUTP, Oregon Green R 488-5-dUTP, "hexas Redt- I 2-dUTP, BODIPY1 630/650-14-dU"I'P, B(3DIPY(g, 650/665-14-dUTP, Alexa Fluork-, 488-5-dUTP, Alexa Fluor`R~ 532-5-dUTP, Alexa Fluork, 568-5-dUTP, Alexa Flaork 594-5-dUTP, Alexa Fluor*) 546-14-dUTP, fluorescein-l2-UTP, tetramethylrhodamine-6-UTP, Texas Redk-5-U"I`P, 695 Cascade BlueV-7-UTP, BODIPY*D FL-14-UTP, BODIPY(K T?vIR-14-UTP, BDDIPY(k, TR-14-UTP, Rhodamin.e Green T~vt 5-UTP, Alexa Fluort 488-5-UTP, Alexa FluorRO, 546-14-UTP (Molecular Probes, Inc. Eugene, OR, USA). Other fluorophores available for post-synthetic attachment include, inter crlia, Alexa Fluort 350, Alexa Fluort 532, Alexa Fluor k 546, Alexa Fluort 568, Alexa Fluork 594, Alexa Fluork, 647, BODIPY
493'543, ;oo BODIPY FL, BODIPY R6G, BODIPY 530/554, BODIPY TMR, BODIPY 558/568, B DIPY 558i568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY
630./650. BODIPY 650. 665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B. Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamiiie 6G, rliodamine green, rhodamine red, tetramethvlrbodarnine, "I'e,xas Red (available from 705 Molecular Probes, Inc., Eugene, OR, USA}, and C~'?, C;y3.5, Cy5.5, and C;17 (Amersham L/tFJ~E, t .... ~ ~.... . k N-1 U'SAc used, such as PerCP-Cy5.5, I'E-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and AI'C-Cy7:
also, PE-Alexa dyes (610, 647, 680) and APC-Alexa dyes. Biotin, or a derivative thereof, may also be used as a label on a detection oligonucleotide. and subsecluently bound by a 171o detectably labe.led av=idin./streptavidin derivative (e.g. phycoerythrin-conj'uoated streptavidin), or a detectably labeled anti-biotin antibody. Digoxigenin may be incorporated as a label and subsequently bound by a d.etectably labeled anti-digoxigenin antibody (e.g.
fluoresceinated anti-dif.ioxigenin). An aminoallyl-dU"1`1' residue may be incorporated into a detection oligonucleotide and subsequently eotipled to aii N-hydroxy succini:mide (NI-IS) 1715 derivitized fluorescent dye, such as those listed stq~ra. In general, any member of a conjugate pair may be incorporated into a detection oiigonucleotide provided that a detectably labeled conjugate partner can be bound to permit detection. As Lrsed herein, the term antibody refers to an antibody molecule of any class, or any subfragment thereof, such as an Fab. Other suitable labels for detection oligonucleotides may include fluorescein 1720 (FAM), digoxigenin, dinitrophenol (DNP), daiisyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6x1-Iis), phosphor-amino acids (e.g. P-tyr, P-ser, P-thr) , or any other suitable label. In one embodiment the following hapten/antibody pairs are used for detection, in which each of the antibodies is derivatized with a detectable label: biotin'a-biotin, digoxigenin/cc-digoxige-nin, dinitrophenol (DNP)/a,-DNP, 5-Carboxytluorescein (FAM)/Ct-725 FAM. As described in schemes below, probes may also be indirectly labeled, especially with a hapten that is then bound by a capture agent, e.g. as disclosed in Holtke et al, U.S.
patent 5.344,757; 5,702,888; and 5,354,657; Huber et al, U.S. patent 5,198,537; Miyoshi, U.S. patent 4,849,336; Misiura and Gait, PCT publication WO 91/17160; and the like.
Many different hapten-capture agent pairs are available for use with the invention.
730 Exemplary, haptens include, biotin, des-biotin and other derivatives, dinitrophenol, dansyl, fluorescein, CY5, and other dyes, digoxigenin, and the like. For biotin, a capture agent may be avidin, streptav-idin, or antibodies. Antibodies mav be used as capture agents for the other haptens (many dye-a.ntibody pairs being commercially available, e.9.
Nlolecular Probes).
735 1001891 In one aspect; pools of probes are provided which preferably have from about I to about 3 bases, allowing for an even and optimized signal for different sequences at degenerate positions. In one embodiment, a concentration adjusted mix of 3-mer building blocks is used in the probe synthesis.

[001901 Probes may be prepared with nucleic acid tag tails instead of being directly labeled.
1740 Tails preferably do not interact with test DNA. These tails may be prepared from natural bases or modified bases such as isoC and isoG that pair only between themseives. If isoC
and isoG nucleotides are used, the sequences may be separately synthesized with a 5' amino-linker, which allows conjul;ation to a 5' carboxy modified linker that is synthesized on to each tagged probe. This allows separately synthesized tag sequences to be combined with 1745 known probes while they are still attached to the column. In one embodiment, 21 tagged sequences are used in combination with 1024 known probes.
100191.1 The tails may be separated from probes by 1-3 or more degenerated bases, abasic sites or other linkers. One approach to minimize interactiora of'taiis and target DNA is to use sequences that are very in.f:requent in the target DNA. For example, 1750 CGCGATATCGCGATAT or CGATCGATCGAT is expected to be infrequent in mammalian genomes. One option is to use probe with tails pre-hybridized with unlabeled tags that would be denaturated and maybe washed away after ligation and before hybridization with labeled tags. Uracil may be used to generate degradable tails;tags and to rem.ove them before running a new cycle instead of using temperature removal;
1755 1001921 In one aspect high-plex multiplex ligation assays of probes are used which are not labeled with fluorescent dyes, thus reducing background and assay costs. For example for 8 colors 4x8=32 different encoding tails may be prepared and 32 probes as a pool may be used in hybridization/ligation. In.the decoding process four cycles each with 8 tags are used.
Thus, each color is used for 4 ta;s used in 4 decoding cycles. After each cycle, tags may be 76o removed or dyes photo bleached, `-I`he process requires that the last set of probes to be decoded has to stay hybridized through 4 decoding cycles.
[00193] In one embodiment, additional properties are included to provide the ability to distinguis~Z different probes using the same color, for example "hrn/stability, degradability by incorporated uracil bases and UDG enzyme, and chemically or photochemically cleavable 765 bonds. A combination of two properties, such as temperature stability directly or after cutting or removing a stabilizer to provide 8 distinct tags for the sanae color; more than one cut type may be used to create 3 or more groups; to execute this 4-8 or 6-12 exposures of the same color may be required, demanding low photo-bleaching conditions such as low intensity light illumination that may be detected by intensified CCDs {ICCDs}.
For example 770 if one property is melting temperature (Tm) and there are 4 tag-oligos or anchors or primers . ,. 'I, ~~"~tl~ o~~t. ~ra~ t ni, a.nothe .,. c ` 4 ,. .
tk) or int, ,._ . _ ~--th a : . , i .

oligo in the first group without stabiiizer. After resolving 4 oligos from the first group by consecutive melting off, the temperature may be reduced to the initial low levei, the 1777 5 stabilizer tnav be cut or removed. and 4 tagged-oligos or anchors or primers can then be differentially melted using the same temperature points as for the first group.
[001941 In one aspect, probe-probe hybrids are stabilized through ligation to another unlabeled oligonucleotide.

Metlivds of se uencirf usirr infers ersed adaptors 1790 1001951 In one aspect, the invention includes a method of determining a nLicleotide sequence of a target polvnucleotide, the method comprising the steps of: (a) generating a plurality of interspersed adaptors within a target polynucleotide, each interspersed adaptor having at least one boundarv with the tarcyet polynucleotide; and (b) determining the ideutitv of at least one nucleotide adjacent to at least one boundary of at least t -o interspersed 785 adaptors, thereby determininp _, a nucleotide sequence of the target polvnucleotide. As is more fully outlined below, the target sequence comprises a position for which sequence information is desired, generally referred to hercin as the "detection position". In general, sequence information (e.g. the identification of the nucleotide at a particular detection position) is desired for a plurality of detection positions. By "plurality" as used herein is 790 meant at least two. In some cases, however, for example in single nucleotide polymorphism (SNP) detection, information may only be desired for a single detection position within any particular tar(yet sequence. As used herein, the base which basepairs with the detection position base in a hybrid is termed the "interrogation position".
100196] An irnportant feature of the invention is the use of interspersed adaptors in target 795 polynucleotide amplicons to acquire sequence information related to the target polynucleotides. A variety of sequencing metbodolociies may be used with interspersed adaptors, including, but not limited to, hybridization-based methods, such as disclosed in Drmanac, U.S. patents 6,464,052; 6,309,824; and 6,401,267; and Drmanac et al, U.S. patent publication 2005/0191656, and sequencing by synthesis meth-ods., e.g. Nyren et al, U.S.
800 patent 6.21 Ct,891; Ronaghi, U.S. patent 6.828, l.00; Ronaghi et al ~
1998}, Science, 281: 36a-355; Balasubramanian, U.S. patent 6,833,246; Quake, U.S. patent 6,911,34-5; Li et al. Proc.
Natl. Acad. Sci., 100: 414-419 (2003); Smith et al, PCI' publication WO
2006/(}7435 1; and ligation-based methods, e.g. Sb.endure et al (2005), Science, 309: 1728-1739, Macevicz, U.S. patent 6,306,597; which references are incorporated by reference.

805 1001971 In one aspect, a method of determining a nucleotide sequence of a tar;~et poly-nu.cleotide in accordance, with the invention comprises the following steps: (a) generating a plurality of target concatemers from the target polynucleotide, each target concatemer comprising multiple copies of a fragment of the target polynucleotide and the plurality of target concateniers including, a number of fragments that substantially covers the 810 target polynncleotide; (b) forming a random array of target concatemers fixed to a surface at a density such that at least a majority oftbe target concatemers are optically resolvable; (c) identifying a sequence of at least a portion of each fragment in each target concatemer; and (d) reconstructing the nucleotide sequence of the target polynucleotide from the identities of the sequences of the portions of fragments of the concatem.ers. Usualiy, ::substantially 815 covers" means that the amount of DNA analyzed contains an equivalent of at least two copies of the target polynucleotide, or in another aspect, at least ten copies, or in another aspect, at least twenty copies, or in another aspect, at least 100 copies.
"Farget polynucleotides may include DNA fragments, including genomic DNA fragments and cDNA fragments, and RNA fragments. Guidance for the step of reconstrticting tar-et 820 polynucleotide sequences can be found in the following references, which are incorporated by reference: Lander et al, Genomics, 2: 231-239 (1988); Vingron et al, J.
Mol. Biol., 235:
1-12 (1994); and like references.
1:001981 In one aspect of the invention, a ligation-based sequencing method may be used as illustrated in Figs. 3A-3E. Many different variations of this sequencing approach may be 825 selected by one of ordinary skill in the art depending on factors, such as, the volume of sequencing desired, the type of labels employed, the type of target polynucleotide amplicons employed and how they are attached to a surface, the desired speed of sequencing operations, signal detection approaches, and the like. The variations shown in Figs. 3A-3E
are only exemplary.
830 1001991 In one aspect of the invention, a labeled probe is able to form a stable hybrid only after ligation to a pairint, probe. Tbe Lise of probe ligation improves data speciticity over standard sequencing by hybridization methods. Probe ligation also has application in position specific base identification {e.g. DNA ends) or in a whole sequence scanning methodology (e.g. all internal overlapping sequences).
835 1002001 `T'o identify sequences at a specific site in the unknown sequence, such as at the ends of the sequence, the labeled probes can be desi(yned to allow ligation to an anchor probe. T llt it~chC a knC3w21 iJdapLOr !4-,:TIt to the end of 'Wn probes can have various numbers of specific and degenerated bases. For example, 2 end 1840 bases can be determined with the probe BBNNNNNN (A = anchor, D= adaptor, G
genomic, B = probe defining bases, N- degenerate bases. ~- label );
AAAAAAAAA.BBNNNNNN*
DDDDDDDDDDDDDDGGGGGGGGGGGGGGGG
[002011 For such a probe structure there are 16 sequ.ence-readinprobes, each consisting of 1845 2 specific bases at the 5-prime end. If all 16 probes are tested, only one would efficiently lioate to the anchor probe and give a strong sif;nal, after removing probes that are not ligated the to anchor probe. Such a positive probe detects two bases at the end of genomic DNA
fragment, with a high specif:icity provided by the strong preference of T4 DNA
ligase for complementary bases close to the ligation site.
1850 f00202] In one aspect of the invention, a sin;Ie stranded target polynucleotide is provided that contains a plurality of interspersed adaptnrs. In Fig. 3A, three interspersed adaptors (3002, 3004, and 3006) are shown, which may be part of an amplicon, such as a concatemer, comprising multiple copies of target polynucleotide (3000). Each interspersed adaptor lias a region (e.g. 3008 and 3012) at each end that has a unique sequence (in this example six such 1855 unique sequences among three interspersed adaptors in all) designed as a biliding site for a corresponding anchor probe, which is an oligonucleotide (which may or may not carry a label) to which a sequencing probe is ligated. Stiich end regions may have lengths in the range of f'rom 6 to 14 nucleotides, and more usually, from 8 to 12 nucleotides. Interspersed adaptors optionally have central region (3010), which may contain additional elements such 1860 as recognition sites for various enzymes (when in double stranded form) or bindin., sites for capture oligonucleotides for immobilizing the target polynucleotide amplicons on a surface, and so on. In one aspect, a sequencing operation with interspersed adaptors (3002-3006) comprises six successive routines of hybridizino anchor probes to each of the different unique anchor probe binding sites. Each such routine comprises a cycle of hybridizing the 865 anchor probe to its end site of its interspersed adaptor. combining with sequencing probes under conditions that permit hybridization of only perfectly matched probes, ligating perfectly matched sequencing probes to juxtaposed anchor prQbes, detecting ligated sequencing probes, identifying ojie or rn~,~re bases adjacent to the anchor probe by the signal generated by the sequencing probe, and removing the sequencing probe and the anchor 870 probe from the target polynucleotide amplicon.
1002031 A further embodiment includes creating a DNA circle of 300-3000 bases in length > adaptors on each st eof tF ' adaptor. In th: ;... ati~- r.=r of two, 20-60 base long sequences, separated by 300-3000 bases is generated. In addition to providing twice the level of sequence data, this method provides valuable mapping 1875 information. Mate pairs can bridge over repeats in de novo sequence assembly, and can also be used to accurately position mutations in repeats lon~er than 20-50 bases in genome re-sequencing. One, or a mating pair of two, -20-50 base sequences can be complemented with probe hybridization or probe-probe ligation data. A. partial set of I;'S
to 1116 of all 5-mers. 6-tners, 7-mers or 8-mers may be scored to provide mapping iraformation for 200-4000 188tt base length fragments. In addition, all probes of a given length (such as all 6-mers) may be scored in 4-16 reaction chambers containing 4-16 sections of the total DNA
array for a given genome. In each chamber '%a to 1/16 of all probes may be scored. After mapping individual DNA fragments all probes can be compiled to provide 100 to 1000 reads per base in overlapped probes in overlapped fragments.
1885 1002041 In one embodiment, the six successive routines are repeated from I to 4 times, preferably from 2 to 3 times, so that nucleotides at different distances from the interspersed adaptor may be identified. In another embodiment, the six successive routines are carried out once, but each cycle of anchor probe hybridization, sequencing probe hybridization, ligating, etc., is repeated from I to 4, or from 2 to 3 times, The former is illustrated in Fig.

890 3A, so that after anchor probe (3015) hybridizes to its binding site in interspersed adaptor (3 )002}, labeled sequencing probes {3016} are added to the reaction mixture under conditions that perinit ligation to aliehor probe (3015) if a perfectly matched duplex is formecl.
1002051 Sequencing probes may have a variety of different structures.
Typica[ly, they contain degenerate sequences and are either directly or indirectly labeled. In the example of 895 Fig. 3A, sequencing probes are directly labeled with, e.g. fluorescent dyes Fl, F2, F3, and F4, which generate signals that are mutually distinguishable, and fluorescent dyes Gl, G2, G3, and G4, which also generate signals that are mutually distinguishable. In this example, since dyes of each set, i.e. F and G, are detected in different cycles, they may be the same dyes. When 8-mer sequencing probes are employed, a set of F-labeled probes for 900 identifying a base irnmediately adjacent to an interspersed adaptor may have the following structure: 3'-FI-NNNNNNNAp, 3'-F2-N';\'NNNNNCp, 3'-F3-NNNN'.\NNGp, 3'-F4-NNNNNNNI'. Here it is assumed that sequence (3000) is in a 5'- 3' orientation from left to right; thus, the F-labeled probes must carry a phosphate Lyroup on their 555' ends, as long as conventional ligase-mediated ligation reactions are used. Likewise. a corresponding set of 905 G-iabc.lcnc1 p~r+~~ .ay have thc- fc'1^wing strtzctare: 3'-ANN'N;T
\*`_\,~N C. I C:N\VNNNNN
. v N: 'm~i49 and for --h~:ir a associated anchor probe must have a 5'-phosphate group. F-labeled probes in successive cycles may have the following structures: 3'-F l-NNNN~NNANp, 3'-F2-NNNNNNCNp, 3'-F3-NNN'riNNGNp, 3'-F4-NNNNNNTN, and 3'-F I-NNNNNANNp, 3'-F2-NNNNNCNNp, 1gto 3'-F3-NNNNNGNNp, 3'-F4-NNNNNTNN, and so on.
1002061 Returning to Fig. 3A, after ligated probe (3018) is identified, it is removed froni. the target polynucleotide amplicon (3020), and the next anchor probe (3022) is hybridized to its respective binding site. G-labeled sequencing probes are hvbridized to the target polynucleotide so that those forming perfectly match duplexes,juxtaposed to the anchor 1915 probe are ligated and identified. This process continues for each anchor probe binding site until the last ligated probe (3028) is identified. 'I'tte whole sequence of cycles is then repeated (3030) using F-labeled sequencing probes and G-labeled sequencing probes that are design to identify a different base adjacent to its respective anchor probe.
1002071 Fig. 3B illustrates a variant of the method of Fig. 3A in which anchor probes are 192o hybridized to their respective binding sites two-at-a-time. Any pair of anchor probes may be employed as Iong as one member of the pair binds to a 3' binding site of an interspersed adaptor and the other member of the pair binds to a 5' binding site of an interspersed adaptor. For directly labeled sequencing probes, as shown, this embodiment requires the use of eiglit distinguishable labels; that is, each of the labels F 1-F4 and G 1-G4 must be 925 distinguishable from one another. In Fig. 3B, anchor probes (3100 and 3102) are hybridized to their respective binding sites in interspersed adaptor (3002), after which a set of sequencing probes (31 04} is added under stringent hybridization conditions.
Probes that form perfectly matched duplexes are ligated, unligated probes are washed away, after which the ligated probes are identified. Cycles of such hybridization, ligation and washing are 930 repeated (3110) with sets of sequencing probes designed to identify bases at different sites adjacent to interspersed adaptor (3C102). The process is then repeated for each interspersed adaptor.
1002081 Fig. 3C illustrates another variant of the embodiment of Fig. 3A, in which sequencing probes for identify bases at every site adjacent to an anchor probe are carried out 935 to completion before an anchor probe for any other interspersed adaptor is used. Briefly, the steps within each dashed box (3200) are carried out for each anchor probe binding site, one at a time; thti-s, each dashed box corresponds to a different anchor probe binding site. Within each box, successive cycles are carried out comprising the steps of hybridizing an anchor probe, ligating secluenc:=r identrfy: -= 'sc-.quencirg, probes.

1940 10[}209] Fig. 3D illustrates an embodiment that employs encoded label.
similar to those used with the encoded adaptors disclosed by Albrecht et al, U.S. patent 6,013,445, which is incorporated herein by reference. The process is similar to that described in Fig. 3C, except that instead of directly labeled sequencizig _probes, such probes are indirectly labeled with oligonucleotide tags. By using such tags, the number of ligation steps can be reduced, since 1945 each sequencin- probe mixture may contain sequences to identify many more than four base.s. For example, non-cross-hybridizing oligonucleotide tags may be selected that correspond to each of sixteen pairs of bases, so that after ligation, ligated sequencing probes may be interrogated with sets of labeled aiiti-tags until each two-base sequence is identified.
Thus, tile sequence of a target polynucleptide adjacent to an anchor probe may be identified 1950 two-at-a-time, or three-at-a-tim.e, or more, using encoded sequencing probes. Going to Fig.
3D, anchor probe (352) is hybridized to anchor binding site (381), after which cncoded sequencing probes are added under conditions that permit only perl:eetly complementary sequencing probes (354) to be ligated to anchor probes (352). After such ligation and washing away of un-ligated sequencinlg probes, labeled anti-tags (358) are successively 1955 hybridized to the oligonucleotide tags of the sequencing probes under stringent conditions so that only labeled anti-tags forming perfectly matched duplexes are detected. A
variety of different labeling schemes may be used witlz the anti-tags. A single label may be used for all anti-tags and each anti-tag may be separately hybridized to the encoded sequencing tags.
Alternatively, sets of anti-tags may be employed to reduce the number of bybridizations and 960 wasliin-s that must be carried out. For example, where each sequencing probe identifies two bases, two sets of four anti-tags each may be applied, wherein each tag in ag;iven set carries a distinct label according to the identitv of one of the two bases identified by the seqaencin"
probe. Likewise, if a sequencing probe identifies three bases, then three sets of four anti-tags each may be used for decoding. Such cycles of decoding may be carried out for each 965 interspersed adaptor, after which additional cycles may be carried out using sequencing probes that identify bases at different sites.
(00210] Fig. 3E illustrates an embodiment similar to that described in Fig.
3B, except that here encoded sequencing probes are employed. Thus, two anchor probes are hybridized to a target polyna.cleotide at a time and the corresponding seqtiencinp- probes are identified bv 970 decoding with labeled anti-tags. As shown, anchor probes (316 and 318) are livbridized to their respective binding sites on interspersed adaptor (3002), after which two sets of encoded seqlien,.im'+ r??'obes (3'17) ~i7't_' added k??IdE',r i,C?ndltit)nc th} tt T?`.,,"'r*t -* iv ~,`:'h r?rC}t7Ã;s fL?rmIrtg oligonucleotide tags of the ligated probes are decoded with labeled anti-tags.
As above, a 1975 variety of schemes are available for decoding the ligated sequencing probes.
1002111 In another aspect, a sequencing method for use with the invention for determining sequences in a plurality of DNA or RNA fra-ment,s comprises the following steps: (a) generating a plurality of polynucleotide molecules each comprising a concatemer of a DNA or RNA fragment; (b) forming a random array of polynucleotide molecules fixed 198o to a surface at a density such that at least a majority of the target coneaterners are optically resolvable; and (c) identifying a sequence of at least a portion of each DNA
or R:~,~A
fragment in resolvable polynucleotides using at least one chemical reaction of an optically detectable reactant. In one embodiment, such optically detectable reactant is an oligonucleotide. In anotlier embodiment, such optically detectable reactant is a nucleoside 1985 triphosphate, e.g. a f7uorescently labeled nLic[eoside triphosphate that may be Lrsed to extend an oligonucleotide, hybridized to a concatemer. In another embodiment, such optically detectable reagent is an oligonucleotide formed by ligating a first and second oli,,onucleotide to form adjacent duplexes on a concatemer. In another embodiment, such chemical reaction is synthesis of DNA or RNA, e.g. by extending a primer hybridized to a 1990 concatemer.
[00212[ In one aspect, parallel sequencing of concatemers of target polynucleolides on a random array is accomplished by combinatorial SBH (cSBH), as disclosed by Drmanac in the above-cited patents. In one aspect, a first and second sets of oligonucleotide probes are provide, wherein each sets has member probes that comprise oligonucleotides having every 995 possible sequence for the defined length of probes in the set. For example, if a set contain.s probes of length six, then it contains 4096 (-4~~) probes. In another aspect, first and second sets of oligonucleotide probes comprise probes having selected nucleotide sequences designed to detect selected sets of target polynucleotides. Sequences are determined by hybridizing one probe or pool ofprobe, hybridizing a second probe or a second pooi of 000 probes, ligating probes that form pcrfectly matched duplexes on their target sequences, identifying those probes that are ligated to obtain sequence information about the target sequence, repeating the steps until all the probes or pools of probes have been hybridized, and determining the nucleotide sequence of the target from the sequence information accumulated during the hybridization and identification steps.
005 10021.31 For sequencing operations, in some erra.bodirzments, the sets may be divided into subs 's tbm 2re used f^-etber in po-? :lisclosed in U.S. 6- 864,052. Probes frorn ti . i .)r in sequence, either as entire sets or as subsets, or pools. In one aspect, lengths of the probes in the first or second sets are in the range of from 5 to 10 nucleotides, and in another aspect, in 2010 the range of froi-ii 5 to 7 nucleotides, so that when ligated they form ligation products with a length in the range of from 10 to 20. and frorn 10 to 14, respective.ly.
1002141 In another aspect, using such tec.bniques. the sequence identity of each attached DNA concatemer may be determined by a"signature" approach. About 50 to 100 or possibly 200 probes are used such that about 25-50 'o or in some applications 10-30% of 2015 attached concatemers will have a full match sequence for each probe. This type of data allows each amplified DNA fragment within a concatemer to be mapped to the reference sequence. For example, by such a process one can. score 64 4-mers (i.e. 25% of all possible 256 4-mers) using 16 hybridization./stripoff cycles in a 4 colors labeling schema. On a 60-70 base fragment amplified in a concatemer about 16 of 64 probes will be positive since there ?020 are 64 possible 4-mers present in a 64 base long sequence (i.e, one quarter of all possible 4-mers). Unrelated 60-70 base fragments will have a very different set of about 16 positive decoding probes. A combination of 16 probes out of 64 probes has a random chance of occurrence in I of every one billion fragments which practically provides a unique sigilature for that concatemer. Scoring 80 probes in 20 cycles and generating 20 positive probes create !025 a signature even more likely to be unique: occurrence by chance is 1 in billion billions.
Previously, a"signature" approach was used to select novel genes from cDNA
libraries. An implementation of a signature approach is to sort obtained intensities of all tested probes and select up to a predefined (expected) number of probes that satisfy the positive probe threshold. These probes will be mapped to sequences of all DNA fragments (sliding window 030 of a longer reference sequence may be used) expected to be present in the array. The sequence that has all or a statistically sufficient number of the selected positive probes is assigned as the sequence of the DNA fragment in the given concatemer. In another approach an expected signal can be defined for all used probes using their pre measured full match and mismatch hybridization/ligation efficiency. In this case a measure similar to the 03-5 correlation factor can be calculated.

100215j A preferred way to score 4-mers is to ligate pairs of probes, for example:Nti.7;,BI313 with BN(7_9). where B is the defined base and N is a de-enerate base. For P-enerating signatures on longer DNA concatemer probes, more unique bases will be used.
For exarnple, a?5% positive rate in a fragment 1000 bases in length would be achieved by N;4_6,BBBB
:z=to a.rrci 131"3N7;64;. N~ote ghOl rr,nger ~ra:, t!r s need the same number of a.bout 60-80 probes (f 3-~

[00216] In one embodiment all probes of a given length (e.g. 4096 N-)4BBBBBBN~_4} or all ligation pairs may be used to determine complete sequence of the DNA in a concatemer. For example, 1024 combinations of N,;_-,B3 and BBN,t,_s) rnayr be scored (256 cycles if 4 colors 2045 are used) to determine seqtiience of DNA fragments of up to about 250 bases, preferably up to about 1 00 bases, [002171 The decoding of sequencing probes with large numbers of Ns may be prepared from multiple syntheses of subsets of sequences at degenerated bases to minimize difference in the efficiency. Each subset is added to the mix at a proper concentration.
Also, some 2050 subsets may have more degenerated positions than others. For exajnple, each of 64 probes from the set N(5_7)BBB may be prepared in 4 different synthesis. One is regular all 5-7 bases to be fully degenerated; second is N4-3(A,T)5BBB; third is NO-2(A,T)(G,C)(A,`I')(G,C)(A,T)BBB, and the fourth is NQ-2(G,C)(A,T)(G,C)(A,T)(G,C)BBB.
1002181 Oligonucleotide preparation from the three specific syntheses is added in to regular to55 synthesis in experimentally determined amounts to increase hybrid ~eneration with target sequences that have in front o#'the BBB sequence an AT rich (e.g. AA"FAT) or (A or 1j and (G or C) alternating sequence (e.g. ACAG'1- or GAGAC). "1'hese sequences are expected to be less efticient in forming a hybrid. All 1024 target sequences can be tested for the efficiency to form hybrid with NO-3NNNNNBBB probes and those types that give the !060 weakest binding may be prepared in aborit 1-1 0 additional synthesis and added to the basic probe preparation.
[00219] In another embodiment, a smaller number of probes is used for a small number of distinct samples; for example, 5-7 positive out of 20 probes (5 cycles using 4 colors) has the capacity to distinguish abotit 10-100 thousand distinct fral;ments 065 1002201 In one aspect, 8-20-rrm.er RCR products are decoded by providing arrays forrned as random distributions of unique 8 to 20 base recognition sequences in the form of DNA
concatemers. The probes are decoded to determine the sequence of the 8-20 base probe region using a ntirnber of'possible methods. In an exemplary method, one half of the sequence is determined by utilizing the hybridization specificity of short probes and the 0:70 ligation specificity of fully matched hybrids. Six to ten bases adjacent to the 12 mer are predefined and act as a support for a 6mer to 10-mer oiigonucleotide. T'his short 6mer will li ;ate at its 3-prime end to one of 4 labeled 6-mers to l0-rners. `I'hese decodin- probes consist of a pool of 4 olicyonucleotides in which each oligonucleotide consists of 4-9 degenerate b-ses and I defined be~~A I'M- o12onucleotide will also bÃ, lab-c'cd with one of' fi_t< ad3e, s. t?r T Wi represented by a fluorescent dye. For example these 5 groups of 4 oligonucleotides and one universal oligonucleotide (Us) can be used in the li~ation assays to sequence first 5 bases of 12-mers: B==each of 4 bases associated with a specific dye or tag at the end:

2080 UUUUt;t.`LJt1.BNN"v`NNNN*
UUUGUUUt;.NBNNNNNN
UUUUULIU[;.NNBNNNNN
UUUt7UUUU.NNNBNNNN
UUUUUUUU.NNNNBNNN

[04221] Six or more bases can be sequenced with additional probe pools. To improve discrimination at positions near the center of the 12-mer the 6-mer oligonucleotide may be positioned further into the 12-mer sequence. This will necessitate the incorporation of degenerate bases into the 3' end of the non-labeled oligonucleotide to accommodate the to9o shift. This is an example of decoding probes for position 6 and 7 in the 12-mer:
CIUUUUUNN.NNNBNNNN
U U1.JUUUNN.NNNN BNNN

!095 [00222] In a similar way the 6 bases from the right side of the 12-mer can be decoded by using a fixed oligonucleotide and _5-prime labeled probes. In the above described system 6 cycles are required to define 6 bases of one side of the 12-mer. With redundant cycle analysis of bases distant to the ligation site this may increase to 7 or 8 cycles. Complete sequencint, of the 12-rner can thus be accomplished with 12-16 cycles of ligation.
too 100223] In one embodiment, the invention provides a method for partial or complete sequencing of arrayed DNA by combining two distinct types of libraries of detector probes.
In this approach one set has probes of the general type N3-aB4.6 (anchors) that are ligated with the first 2 or 3 or 4 probes:/probe pools from the set BN(,-g, NBN5_-, N2F3N4-6. and N3BN;.;. In an exemplary method. 1-4 4-mers or more are hybridized to 5-mer anchors to 105 obtain I or 2 anchors per DNA for about ~0%-80% of the molecules. In one ernbodinsent, the positive anchor is determined by mixing specitic probes with distinct hybrid stability (maybe different number of Ns in addition). Anchors may be also tagged to determine which anchor from the pool is bybridired to a spot. Tags, as additional 1n)NA
seoments, mav be used tc~~ a~_F as a detectior For 21 to EEEI/EEEENNNAA.A.AA and FFFFFFFFNNNCCCCC probes can be after hybridization or hybridization and ligation differentially removed with two corresponding displacers:
EE-EEEEEENNNNN and F1;Fl`laF1=FNNNNNNNNwhere the second is more efficient. In another embodiment, separate cycles may be used to determine which anchor is positive. For this purpose anchors labeled or tagged with multiple colors may be ligated to unlabeled N7-2115 N10 supporter oligonucleotides.

1002241 Tbe BNNNNNNNN probe is then hybridized with 4 coiors corresponding to bases. A discriminative wash or dispiacement by complement to the tag is used to read which of two scored bases is associated to an anchor if two anchors are positive in one DNA. Thus, two 7-10 base sequences can be scored at the same time. 2-4 cyc(es can be 2120 used to extend to a 4-6 base anchor for an additional 2-4 base run of 16 different anchors per each array (32-64 physical cycles if 4 colors are used) to determine about 16 possible 8-mers (r-100 bases total) per each fragment. This is sufficient to map it to the reference probability that a 100-mer will have a set of 10 8-mers is less than 1 in trillion trillions; (l0e-28). By combining data from different anchors scored in parallel on the same fragment in another 2125 array complete sequence of that fragment and by extension to entire genomes may be generated from overlapping 7-10-mers.
1002251 In one aspect, the invention provides methods for tagging probes with DNA tags for larger multiplex of decoding or sequence determination probes. Instead of a direct label, the probes can be tagged with different oiigonucleotide seqtjences made of natural bases or '. 13o new synthetic bases (such as isoG and isoC). Tags can be designed to have very precise binding efficiency with their anti-tags using different oligonu.cl~:otide lengths (about 6-24 bases) and/or sequence including GC coDteDt. For example 4 different tags may be designed that can be recognized with specific anti-tags in 4 consecutive cycles or in one hybridization cycle follozved by a discriminative u-ash. In the discriminative wash, the initial signal is 135 reduced to 95-99%, 30-40%, 10-20% and 0-5% for each tag, respectively. In this case by obtaining two images 4 measurements are obtained assuming that probes with different tags will rarely hybridize to the same dot. Another benefrt of having many different tags even if thev are consecutively decoded (or 2-16 at a time labeled with 2-16 distinct colors) is the ability to use a large number of indiiidually recognizable probes in one assay reaction. This 140 way a 4-64 times longer assay time (that may provide more specific or stronger signal) may be affordable if the probes are decoded in short incubation and reniova1 reactions.

1002261 `1`hl- dccodir-L- pr.-z t-.= reqtiire5 .~ ,. o1'48-96 - decodin~_, pr<;bes. 'l'hese Eols by f7t,zprophores, each. having different emission spectra. Using a 20x objective, each 6 mm x 6 2Ã45 mm array rnay require roughly 30 inia;cs for full coverage by using a 10 mega pixel camera.
Fach 1 micrometer array area is read by about 8 pixels, Each image can be acquired in 250 milliseconds: 1.50 ms for exposure and I00 ms to move the stage. Using this fast acquisition it will take -,-7.3 seconds to image each array, or 12 minutes to image the complete set of 96 arrays on each substrate.
2150 [002271 In one embodiment of an imaging system, a high image acquisition rate is achieved by using four ten-megapixel cameras, each imaging the emission spectra of a diftcrent fluorophore. The cameras are coupled to the microscope through a series of dichroic beam splitters. "I'he autofocus routine, which takes extra time, runs only if an acquired image is out of focus. It will then store the Z axis position ini'orrnation to be used upon return to that 2155 section of that array during the next imaging cycle. By mapping the autofocus position for eacb location on the substrate we will drastically reduce the time required for image acquisition.
1002281 Typically, each array requires about 12-24 cycles to decode. Each cycle consists of a hybridization, wash, array imaging, and strip-off step. These steps, in their respective t 160 orders, may take for the above example 5, 2, 12, and 5 minutes each, for a total of 24 minutes each cycle, or roughly 5-10 hours for each array, if the operations are performed linearly. The time to decode each array can be reduced by a factor of two by allowing the system to image constantly. To accomplish this, the iniaging of two separate substrates on each microscope is staggered, i.e., while one substrate is being reacted, the other substrate is ? I 65 imaged.

[002291 An exemplary decoding cycle using cSBi-I includes the followinc, steps: (i) set temperature of array to hybridization temperature (usually in the range 5-25"C); (ii) use robot pipetter to pre mix a small amount of decoding probe with the appropriate amount of hybridization buffer; (iii) pipette mixed reagents into hybridization chamber;
(iv) hybridize !t 7o for predetermined time;. (v) drain reagents from chamber using pump (syringe or other); (vi) add a buffer to wash mismatches of non-hybrids; {vii) adjust chamber temperature to appropriate wash temp (about 10-4Ã1 "C); (viii) drain chamber; (ix) add more wash buffer if needed to improve imaging; (x) imau.e each array, preferably with a mid power (20x) microscope objective optically coupled to a high pixel count high sensitivity CCD camera, a~s or cam.eras; plate stage moves chambers (or perhaps flow-cells with input funnels) over ', *:, ', ctflVe-t'F.r~cs assC ~~~'!',.' moves Ltt"I" C;ha1Tlbera "FttS, n b ed t b-5 simultaneously, thus decreasing iznage acquisition time; arravs can be imaged in sections or whole, depending on arrayiima.ge, sizeipixel density; sections can be assembled by aligning 2180 images using statistically significant empty regions pre4coded. onto substrate (during active site creation) or can be made using a multi step nano-printing technique, for example sites (grid of activated sites) can be printed using specific capture probe, leaving empty regions in the grid; then print a different pattern or capture probe in that region using separate print head; (xi) drain chamber and replace with probe strip buffer (or use the buffer already, 2185 loaded) then heat chamber to probe strip off temperature (60-90 C); high pH buffer may be used in the strip-off step to reduce stripoff temperature; wait for the specified time; (xii) remove baffer; (xiii) start next cycle with next decoding probe pool in set.

Co-nbiriatoriat probe ligation for sequencing by hybridization [00230] In a preferred aspect of the invention, information on the sequence of a target >_ t 9o polynucleotide is obtained through a sequencing by liybridization method which utilizes combinatorial probe ligation. In this aspect of the invention, two complete, universal sets of short probes are exposed to target DNA in the presence of DNA ligase (R.
Drrnanac, l;S
patent 6,401,267, 2002). Typically one probe set is attached to a solid support such as a glass slide, while the other set, labeled with fluorophores, is mobile in solution.
When attached ! 195 and labeled probes hybridize to the target at precisely adjacent positions, they are ligated, generating a long, labeled probe that is covalently linked to the slidc surface. A positive signal at a given position indicates the presence of a sequence within the target that complements the two probes that were combined to generate the signal.
1002311 In a preferred embodiment a universal sequencing uencin~ chip, such as the HYChip ~'ki slide 200 developed by Complete Genomics, is used in the combinatorial sequencing by hybridization methods of the present invention. In one embodiment, each HvChipJIM comprises a regular microscope glass slide containing eight replica arrays of attached 6-mers, allowing analysis using a complete set of over four million 11-rner probes per sample usinT 4096 arrayed 6-rners and 1024 labeled 3-rrier probes. In a preferred embodiment, the sequencing method 20 -5 utilizing the HyChip]'M system is used to sequence mixtures of separate, unrelated DNA
fragments.
[00232] DNA samples for use with the sequencing methods of the present invention can be prepared by PCR.
1002331 in a preferred aspect, the invention provides an array of millions of individual at Z 61e6F3d.CY of about one spot per square micron. These poly=nucleotide molecules scrve as templates for hybridization and lWation of fluorescent-tagged probe pools. In one embodiment, probe pools are rnixed with DNA ligase and presented to the random array. When probes hybridize to adjacent sites on a taroet fra~,=ment, they- are Iipted to~,~etl"ier, forming a stable hy=brid. A
2215 sensitive mega pixel CCD camera with advanced optics can be used to simultaneously detect millions of these individual hybridization/ligation events on the entire array. Once signals from the first pool pair are dctected, the probes are removed and successive ligation cycles are used to test different probe combinations. In preferred aspects of the in~-ention, a 3.2 x 3.2 mm array will have the capacity to hold 10 million fragments, or approximately 1-10 222o billion DNA bases.

Cornbitratorial labeling using labeled tags [00234] In one aspect, a single hybridization/ligation cycle can be used to test all 16 possible probes by using 16 fluorescent colors. Such a test may also be accomplished using methodologies to create f]Liorescent signatures from fewer fluorescent colors.
In fluorescent !225 in-situ hybridization (FISH) chromosomal "painting" combinations of fluorescent probes can be utilized to create new fluorescent signatures for that combination of probes. For example, combinations of two probes from a set of 4 can create 10 possible signature fluorescent signals, 5 can create 15, 6 can create 21 and so on. Therefore, in a single hybridization cycle it would be possible to distinguish which one of 16 probes was '230 hybridized to the anchor probe.
[00235] Alternatively, if one of the BBNNNNNN probes was left unlabeled (and inferred by lack of signals for all other probes), 5 colors would be sufficient to label all of the remaining 15 dinucleotides. Four colors may be used to label 4 probes that read a single base, or 8 probes (out of all 16 needed probes) to read two bases. In this latter case all 16 235 probes could be scored in two cycles (see below). Thus, a 5 or 6 color system may- be much easier to implement than 16 colors required by non-combinatorial labeling.
1002361 For efficient combinatorial labeling, 2-mer probes may be prepared with a tail sequence containing tan binding sites. Tail sequences can be corn.birtatorially desil;ned for binding 2 out of 5 (or 6) labeled oligonucleotide tags or 16 tags with one or two fluorescent 240 dyes can be synthesized for each oftbe 16 tails. Use of'labeled tags instead of directly labeled probes has additional aÃIvantages. Testing all 16 BBNNNNNN probes would require about 1024-fold more probe (assuming low discriinin.ation at pctiitions furtl?,_,r from the ;. ,._ ~ . For j ha~

concentration withiix a probe mix of BBNNNNNN, the mix should need to be at 10214 M.
2245 Since labeled probes are much costlier to synthesize than unlabeled probes, the unlabeled probes could be detected with a tail sequence, with the labeled tag probe used at a low concentration since it -xaay be perfectly complementary to the tail sequence.
Additionally, using unlabeled tailed probes woLild be advantageous in maintaining a lower background becatise the fluorophore would be at low concentration. An overall I 00-fold cost reduction is 2250 expected by using 6 labeled tags (without degenerate bases) instead of the equivalent 1024 labeled probes.

1002371 Tags also provide an efficient option to use only 4 colors to read all dinucleotides in a single ligation reaction. In such an embodiment, two sets of 4 distinct tags may be designed for decoding 8 2-mers each. All 16 2-zners can be decoded in two decoding 2255 cycles. This strategy can be expanded to use the same 4 colors for reading 2 bases on each end of an adaptor. In this case, 4 groups of 4 tags may be used in. 4 decoding steps for each ligation cycle that reads 4 bases. Performing miiltiple decoding cycles instead of multiple ligation cycles is less expensive (less enzyme is used), and ligation cycles may be extended for longer time, with lower probe concentration, to reduce mismatch ligation.
2260 [002381 Tags may also be designed to ininimize interference witll tlle analyzed DNA., for example by using isoC and isoG base pairs that do not pair with natural bases.
Another option is to use standard DNA chemistry but design sequences that are very infrequent in the human genome. Yet another option is to use a probe with tails pre-hybridized with unlabeled tags that would be removed after ligation and before hybridization with labeled tags.

>.265 Expatrrling the number of bases that can be decoded 1002391 To read further than 2 nucleotides from the anchor probe can in some aspects of the invention utilize additional rounds of probe-anchor ligation, with removal of the anchorf'label probe from the target prior to the initiation of the next cvcle.
The ligated probe-anchor can be removed usina a number of rnethads known in the art, including by heating Z7, 270 or by temperature or lic-lht cleavable bonds in the anchor probe, such that the anchor is fragmented and destabilized in tbe heating step. Since the bases to be seqtEenced are now 3 and 4 bases from the adaptor, rnodifications need to be made to the anchor probe or labeled probe. ln the case of-the anchor probe, it can in one embodiment of the invention be prepared with 2 additional degenerate bases at the ligation end. To ensure that the efficiency 275 ot'the subsequent ligation is mai.ntained, in one embodiment the anchor is constrLicted _ Wo s, ~f~i.:... . , tl~ 1 sequencing probe can be prepared with two degenerate bases at the ligating end in the manner of: NNBBNNNN-tag. In another aspect of the invention, the assay may be designed to read an additional 2 bases using 16 anchor probes.
2280 f002401 1be specificity of probe-ancbor ligation is very high because only 2-4 bases around the lioation site are tested. ri'be avera,le discrimination for these bases is 50-1 00 fold. Some mismatehes such as GT are considerably stronger, havinsw discriminations of only 5-20 fold.
In an embodiment of the invention, software is provided that can take the differences in discrimination of certain mismatches into account.
2285 1002411 In an aspect of the invention, each probe, anchor and tag is optimized (for example, by concentration, number of degenerated bases, sequence and length of tags) to maximally equalize full match signals. Overlapped and shifted pairs of probes and anchors may be designed in one embodiment of the invention to read each base 2-3 times to increase base calling accuracy.

2290 1002421 The insertion of additional internal adaptors with anchor regions at precise short distances expands the sequencing capability of bases at defined positions in the genomic fragment. For example, having the original plus 2 additional adaptors spaced 8 bases apart allows the determination of'20 continuous bases in 1.0 cycles. by reading 4 bases from 5 consecutive adaptor ends.
?2J5 Initial adaptor First 8 bases Adaptor 2 2'", 8 bases Adaptor 3 Addrttonal -20(1 bases DDDDDDDDDTSGGGGGGGGDDDDDDDDDDGGGGGGGGDDDC]DDDDDDGGGGGGGGGGG
AAAAAAA.BBNNNNNN-taik AAAAAAA.BBNNNNNN-tail AA,AAAAA.BBNNNNNN-tail AAAAAAA_NNBBNNNN-tail AAAAAAA.NNBBNNNN-tail AAAAAAA.NNBBNNNN-tail '.300 tail-NNNNBBNN.AAAAAAA tail-NN~INBBNN.AAAAAAA
tai[-NNNNNNBB.AAAAAAA tai[-NNNNNNBB.AAAAAAA
D= adaptor, G= genomic DNTA anchor, B==- specified probe base, N- degenerate probe base.
[00243] Multiple adaptors also provide the opportunity to further increase the reading capacity and to be able to determi.ne more than 2 bases per cycle. In one embodiment, 4-12 305 bases are identified per cycie. In another embodiment, 4-8 bases are identified per cycle. In yet another embodiment, 12-16 or more bases are determined per cycle.
[00244] In one embodiment, 3 adaptors are positioned 1. 2 bases apart, allowing for 3 )0 bases of continuous sequence to be obtained by reading 6 bases at each of 5 ends. In another embodiment, a total of 4 adaptors and reading 16 bases betkveen two adaptors ~-Yenerates a 110 continuous sequence of 56 bases in 28 cycles. In other embodiments, two (initial plus one additional) adaptors separated by 16 bases to read 24 bases are used.
1002451 In one einbodirient, multiple bases are identified per cycle by simultaneously hvbridirincy probes t(. 'ipa~ or all anchor site:s witi:~ the saaic set of 16 dinucleotide probes used at each anchor site but read each anchor site in.dependently. In one embodiment, this 2315 simultaneous probe Iigation is achieved by designing anchors with different melting ternperatures and measuring color intensities at multiple predefined temperatures.
1002461 In another embodiment, multiple adaptors are used for cyclical primer extensioii to provide longer reads with fewer cycles from each individual primer.
[002471 In one einbodiment, mapping information can be obtained by scoring a sLifficient 2320 number of short sequences distributed over the entire DNA fragment without any positional information or from a smaller number of short sequences at precise locations.
A variant of this process is referred to as "hybridization signatLire" where expected and obsenied intensities are compared. In another embodiment, the short sequences may be designed to provide localized (intermittent or continuous) sequence information. Three examples of such 1-325 short sequences may be represented schematically as follows:
a. (X)aBB(X)bBB(X)cBB(X)dBB(X)eBB(X)f...
b 1. BBX6BBX4BBX6BBX4BBXa.. ..
b2. B I 6Xa 1002481 The number of oligonuclcotide sequences needed for complete mapping !330 information depends on the size of the target sequence, the size of the DNA fragments used and on the complexity of the source DNA. For human and other similarly complex genomes about 5 positive 8-mers or 10 positive 6-mers may be sufficient for 100 base DNA
fragments. To score one positive 8-mer in 2 cycles, about 10 cycles total can be tised by employing 3-fold more cycles than anchor sequencing. ln one embodiment, this process 335 does not utilize insertion of two anchors and may be done without enzyme using direct hybridization. In such an embodiment, 3000 8-mers can be utilized.
[002491 In one embodiment the same set of probes may be used in diflerent group combinations (combinatorial pooling) to decode which probe from the pool of probes with identical labels is positive. For example, all 3000 probes labeled with 300 distinct labels may 340 be scored in two reactions by having 5 probes labeled with the same probe corn:bination. In addition to 6 true positives, some other 30 or rnore pool-related false positives will be found in these two reaction..s. By perforrrsing another two hybridization cycles where probes will be grouped differently, only true positive probes will be decoded since they are sha.red positives between two data sets and with less than one false positive probe being shared. Finding 345 positive probes may be performed by using the lower of the two scores for each probe. For true. pnc,;,.Y-_~^robGs the score i~ to be ~z,21F at least :, _ _helps reduce the number of cycles or number of required labels and may provide enough power for many applications withatit the need to use combinatorial labeling.
2350 [00250] In another embodiment, highly overlapped sets of fragments analyzed in the form of 2-16 subsets on different subarrays with different subsets ofprobe.s provides a large amount of mapping information. For example 250 base fragments starting at every base on average can be analyzed as 2-16 subsets with 2-16 different subsets of probes.
DNA
fragments that are shifted only 2-26 bases will be analyzed with a few if not all used probe 2355 subsets providing unique chromosomal identification with at least one probe subset.
[002511 Tvpically, twenty specific bases will provide the information necessary for most unique sequences. In one embodiment, this inforrriation can be obtained with two anchors in cycles with. 256 tags for reading 5x4 bases, or 3 cycies for 24 bases by reading 8 bases per cycle (512 tagging combinations). In another embodiment, 3 cycles x 6 bases=l8 bases 2360 (5x3+3 at a distance of 20-30 bases), and in yet another embodiment 4 times less tags for 3-mers, may need 3 anchors (3x6 + 3-3 bases).
1002521 In one aspect, a high capacity DNA array platform can be used to analyze 100 patient or other DNA samples simultaneously. In the direct hybridization (or combinatorial ligation) approach of mapping, only a subset of probes is used and does not provide tag 3365 sequence automatically. For 4-base tags all 256 probes (e.g.
NxUxBBBB(JxNx) may be used for mapping or as additional probes. If these probes are also used for mapping multiple sets of 256 shifted probes may be needed to identify the tag sequence.
1002531 In one aspect, 5-6 colors are used to decode all 16 dinucleotides and read 2-12 bases in one decoding cycle. In one embodiment, a set of 4 tabs is used; in another 370 embodirnent, the set is expanded to 6 tap. Multiple decoding cycles alone or in combination with anchors with different melting temperatures can be used to increase the ntrmber of bases that can be read in a single decodin.lg cycle.
1002541 In one aspect, 4 bases per ligation cycle are read by testing 2 bases on each end of an adaptor and by using two corresponding anchors. Both types of probes B2N6-tail and tail-375 N6B2 may be used simultaneously. Each probe type may have unique tails and a matching set of 6 unique tags. Two decoding cycles, using two sets of 6 tags, would identify 4 bases.
In I 1 ligation cycles 42 continuous and 2 redundant bases would be determined. rl,o read a mate-pair of 42+18=60 bases, 15 ligation cycles would be required.
1002551 In another aspect, 8 bases are read per ligation eycle. A total of 4 anchors may be 38o used "each oft~v<> sideE ol 4iy ad rC Pi- 1*c~ an' -may be `'~~: ~st same as ~,~ ~

o g}~I~.>Z~, ! hL24, 3rl t . Ee . s . t Of r c1 Because an additional 2 anchors may be used for the second adaptor, additional information is needed to discriminate which of the two positive 2-mers belon=s tc~ which anchor/adaptor end. This can be achieved by designing the two anchors for the second adaptor with higher 2385 melting temperatures (Tm). Thus, schematically, the 4 anchors are:

adaptor I adaptor2 .,.GGGGDDDDDDDDDDDDDDDDGGGGGGGGGGGGDDDDDDDDDDDDDDDDDDDDDDDDDDGG
GGG...

IJ=== adaptor bases, tJ === genomic bcrses. A === anchor bases, 1002561 After two standard cycles of decoding and imaging of 5-6 dyes, a stritigent wash can be applied that removes low "I`m anchors and the tailed probes that are ligated to them.
2395 but does not affect high "I'm anchors. By repeating two cycles of tao binding and measurin.w=
fluorescence, the Iluorescence signals specific to the second adaptor with longer (higher Tm) anchors is deterniined. The difference between the first and second set of measurements gives the signal produced by 2-mers corresponding to the first adaptor. A
strip-off wash at even higher temperature would remove higher Tm anchors and free DNA for the next >_400 ligation cycle. Higher Tm anchors may be photo, chemically or temperature cleavable for easy strip-off. To read more bases the process can be repeated 3 times to read 24 bases surroundino two adaptors, or 6 times to read 48 bases surrounding 4 adaptors.
"1'o read the remaining 12 bases for the lifth adaptor, 3 additional cycles may be required.
In these 3 cycles, repeat sequencing of 12 previously sequenced bases with the same or shifted anchor-!405 probe pair may also serve as a control of data quality. In total, 9 ligation cycles and 36 decoding cycles can be used to determine 72 bases (60 unique and 1.2 repeated).
1002571 In another aspect, 12 bases are read per cycle by expanding the process from 2 to 3 levels, providing a read of 12 bases {3x2x2) per ligation cycle. Si--nilarlv, 72 bases (60 unique and 12 repeated) can be determined in jLast 61il;atiort cycles. The Ttn approach can be !41tt used in many other configurations with an increased number of anchors that can be differentially removed one by one. The lcey advanta~e of this approach is that in one ligation reaction, probes of one type are ligated to 3 different anchors.
1002581 In another aspect, 8 bases are read in one ligation cycle without usinf; T`m differentiation of anchors. To achieve this, the anchor probes are designed to read 2 bases 415 simultaneously with a 2 base read by the non-aricltor probes. Two such pairs cara be analyzed in one ligation cycles reading a total of 8 bases per cycle as follows.

~Ã)DDDDDDt >C~t~GGt~t~~~C;`GCt r `)D

ta.il-AAAAAABB.BBNNvNNNJr1fL TA[L-NNNNNNBB.BBAAAr~f~A-tail 2420 (cycle 1) ~ ~
tail-At1ANNNNBB.BBNNNNNN-TAIi. TA[L-NNNNNN BB. BBNNNNAAA-tail (cycle 2) Ãail-NNNNNNBR.BBA.4AAAA-'rAlt. 't`ATL-t1Af~AAABi1.BBNNNNNN-tail (cN,cie 3) D= ar7aptot- bases, G=: genorrric bases. A = anchor bases, R-- s=pecifedprÃzbe hures, N-degerrerafe probe bases [00259) Decoding, would be performed in four cycles having 4 sets of tags specific for each of 4 tail groups. Interestingly, this approach may provide 44+20=64 bases using -5 adaptors 21430 (8+4x12-~-8) in 8 ligation cycles without Lienerating anv redundant base reads. Readina 16 instead of 12 bases between two adaptors and a total of 80 bases using 5 adaptors is a natural progression for this system. The main new development that may be required is to implement a stabilization process for the probe-anchor ligation product that is compatible with the encoding tail present at the anchor probe.
2435 [002601 These processes coupled with inserting 1-2 additional adaptors 12 bases apart, can increase parallel reading per ligation cycle from 2 to 8 or even 12 bases in.just 6-15 ligation cycles. In a further embodiment, 16 bases are read between neighboring adaptors, allowing the use ofonl}- the initial + 2 inserted adaptors, leading to the ability to determine 40 (2x 16+8) bases of continuous sequence.
>440 Multiplex prabe-ancfiar ligation assay [00261] In one aspect, probe sets comprising 16 probes of the structure BBNI~~NNNN-tail in which the tail is approximately 15 to 20 bases in length and a complementary tag sequence to the tail labeled with fluorophores are prepared. Tails and tags are designed to minimize interference with the analyzed DNA. In one embodiment, tail and tag sequences are 445 prepared from iso-c and iso-g nucleotides to prevent the tag sequence from interacting with the template DNA.

[002621 It is possible to test the efficiency ofdifferent BI3NNNN;=rTN-tail probes with different tail and tag sequences. Sixteen tail sequences may be required, but only eight of the 16 probes (with 16 different tails) may be analyzed in, each decoding cycle since the 450 maximum capacity of the 4-color mixing is 10 possible combinations of two (not including a null signal as a possible probe indicator). Each tail sequence may have the capacity to bind two tags, and each tag in this design may only have one fluorophore attached.
An initial desip ,rt of-a set of 4 taL=s. one for each color may be performed. The cornplementarv sequences of these tags may be combined to create 8 tails (out of a total of 10 possible 2455 combinations). The remaining 8 of the 16 tails may also require an additional set of 4 tags but they can carry the same flLiÃ3ropbores as used for the first set of 4 tags.
1[}02631 In one aspect. probes may be prepared with a single tluorophore (e.g., "fAMR.A) to determine the relative strengths of the different tag combinations (i.e.
hybrid strengths).
Once this inforniation is obtained it is possible to match the fluorophores to the tags to 246o normalize intensities. A single fluorophore set of tags can also be used to determine the relative efficiencies of the BBNNNNNN region of the probe with a common tail structure.
Once these parameters have been determined, a set of 16 BBNNNNNN-tail probes can be prepared. This probe set may be used to hybridize to RCR products derived from the PCR
and synthetic target circles or even complex genomic samples.
?465 1002641 In one embodiment, arraved RCR targets are first hybridized with an adaptor probe to deterniine the DNB locations and relative intensities. This probe is removed using standard techniques, such as by raising the temperature, and a second set ol' probes can then be hybridized to the arrav. The second probe set contains an anchor probe and BBNNNNNN-tail probes in a ligation mix. "1'he reaction proceeds for a su1-3icient length of !470 time, preferably for about 30 minutes, and the unlil;ated, unhybridized probes are then washed away. The next addition to the chamber can include the 4 tag probes that hybridize to the tails of ligated and hybridized BBNNN-NNN probes. This hybridization can in some embodiments be as short as 5 minutes to achieve high signal intensities. The chamber is again washed and imaging occurs at the desired wavelengths. I'h.e chamber then undergoes .475 heating to remove the tags but maintain the anchor-BBNNNNNN-tail probes in the hybrid.
The second group of 4 tags can then be hybridized to score the presence of the second group of 8 BB?~~INNN probes. The level of discriminatiori between the matching BBNNNNINN
probe and the other 15 mismatch BBNNNNNN probes can be determined through the level and combinations of signal intensity.
480 1002651 In one embodiment, to establish a probe-anchor ligation assay, a probe is provided, for example a probe of strL-cture AANNNNNN, to generate enoLrgh of a signal for an AATA'I'ANN DNA spot with a low AG for the 'I'A"I'A sequence. If the signal tEor the optinial condition is low for some DNA sequences, matchin;g probes can be prepared independe.ntly and added into the mix to selectively boost concentrations only for these probes. if ?(1 485 sequences out of256 at the first 4 degenerated positions have to be ad}'usted, 16x'20 additional probes can be prepared.

[00266[ In one embodiment, development and testing 16 probes for reading 2-base sequences from the other side of the genomic segment between two adaptors is accomplished. Ta.il and degenerated bases for these probes may be at the 5' end, e.g. Taii-24go NN'~N7Nf3B.

1002671 In one aspect of the invention, the number of dves that can be differentiated is maximized by using multiple specific excitation patterns and a maximal number of filters for each excitation pattern. For example, 2-4 excitations, each witb. 4 different v,?ave lengths (total of 16 wave lengths) can be used in combination with 8-16 filters for each excitation.
2495 Algorithm and software is used to analyze intensity patterns and deduce the amount of signal from each of the 8-24 dyes.

[002681 In one embodinlent, direct labeling with dyes is combined with indirect labeling using haptens (such as biotin) to specifically stain multiple probes. Directly attached dyes may be photo-bleached or differences in the intensity may be calculated before and after ?500 staining.

[00269] In one embodiment, the number of color labels available for use is expanded by light or chemical de-blocking of quenchers or chemical modifications that shift absorption of the given dye. Color intensities are measured before and after de-blocking treatment. After the first imaging is done the dye may be photo-bleached before an increase of signal for tlle '505 given wave length is measLared. With multiple types of quenchers or modifiers (3-4-6) and 8 colors a total of 24-48 non combinatorial labels can be generated.
Combinatorial labeling with 2 out of 24-48 labels gives a potential of276-1128 two-label combinations.
[00270] Long stable anchors provide can improve probe hybridization and ligation to different targets. In one emboditnent. the number of degenerate bases is increased to 510 minirriize the influence of target sequences that form unstable hybrids such as 5'"I'ATA3'.
This may increase the stability of probe/target hybrid but a probe that does not have a full match at the first 2-4 positions close to the ligation site may hybridize to the target and prevent ligation. To minimize this negative influence, one einbodimertt provides a higher starting temperature and/or temperature cycling to increase the number of ligatable probes 515 hybridized next to the anchor.

Sequencing using prirtrer extension [00271[ End sequencing may be performed l:rom one anchor;=primer end by many consectztiicycles of ;ingle base extension using specifically labeled nucleotides. In on_ tep inwbicb .,.. K

2520 extension. Multiple adaptors provide increased flexibility in this process. In one embodiment, 2-6 or more bases are read by sinale base primer extension using shifted primers in consecutive reactions, Multiple simultaneous shifted 04-1 or 1.-3-I
primer ti-ames on one adaptor or single frazne on multiple adaptors or both may be used.
1002721 In one embodinient, using the initial pltis 3 addit-onal anchors provides 4 primers.
252-i By reading 4 bases of each primer, 16 bases are determined in 16 cycles using 4 standard colors, which can be accomplished without combinatorial labeling or tagging.
In this embodiment, the primer extension does not have degenerate bases on the labeled component, thus reducing the concentration of dyes tised. Because 16 bases may not be sufficient for mapping, 4 primers x 5-6 bases of extension in 20-24 cycles can be used.
21530 1002731 Multiplex primer extension is possible by discriminative removal of the primers.
Several different methods may be used for such removal based on factors including: primer length, GC content, base or backbone modifications such as LNA or PNA, uracil incorporation, or light sensitive linkage between selected bases. Two to eight stability levels in one group may be designed. Also 2 to 4 distinct groups that may have different stabilizers ?535 or protectors can be used. By applying these labeling methods, 20-24 bases may be determined in as few as 3-5 enzymatic cycles. In another embodiment, a primer protection assay for multiplex primer extension one base at a time is used. In such an embodiment, the primer, for example UUlJUL3UUNNN. used for the fourth extension provides enough signal because mismatches at NNN can occupy over 50% or over 90% of the target and would not !54o be efficiently extended. Primer with higher specificity may be created by ligating UUtJGUUU.UUUNNN or UUtJ[.;U UIJ.CTNNNNN.
1002741 In one aspect, in order to be able to sequence on each side of the anchor, the attached ssDNA may be converted in dsD':_~A using the attached primer and removal of the original strand or primer invasion techniques. One approach to remove the original strand is 545 to incorporate in inserted adaptor binding site for a restriction enzyme that cuts only one strand. The fragmented strand would then be denatured and washed away.
[002751 For performing consecutive or overlapped frames or reading 2-3 bases a different anchor and or probe design may be used. For example:
Cycle 1: liULL'UI, iliL`L:UL;.BBNNNNNN
5 0 Cycle 2: L~~L`UUUL`LUL'NN.BBNNNNNN or UUC `L` I~~ li UI; Ut: L.NN BBNNNN
Cycle 3: t'L.EUC;I.TUi'L'tNN.NNBBN:~'"NN
~~
2 i~t -P-del., ted basc and 76 1002761 Anchors that have degenerated bases may be designed in two parts to assure preferential binding of anchors that have matching bases at degenerated positions.
Overlapped or shifted frames may be used to read each base multiple times in the same target. "1`wo examples for multiple reading of the first four bases after the anchor are 2560 presented below:
UUUUUUUUI.liU.UBBNNNNN
UUUUUUUUUUU.BBNNNNNN
UUUUUUUUUUN.BBNNNNNN
256i UUUUUUUUUUU.NNBBNNNN
UUUUUUUUUNN.BBNNrNNNN
UUUUUUUUUUN.BBNNNNNN
Where U represents common pre-defined bases.. B a specified base an.d N a degenerate base.
2570 The ligation site is indicated with a period (.}

Detection instrumentation 1002771 In one aspect of the invention, hardware is provided to allow detection of the ligation and hybridization events of the sequencing methods. In one embodiment, the ;575 system hardware comprises three major components; the illumination system, the reaction chamber, and the detector system. The detection instrument can include several features such as: adjustable laser power, electronic shutter, auto focus, and operating software.
[002781 Signals from single molecules on random arrays made in accordance with the invention can generated and detected by a number of detection systems, including, but not ~580 limited to, scanning electron i~ieroscopy, near field scanning optical microsc-opSt (NSOM), total internal reflection fluorescence microscopy (TIRFM), and the like.
Abundant guidance is found in the literature for applying such techniques for analyzing and detecting nanoscale structures on surfaces, as evidenced by the following references that are incorporated by reference: Reimer et al, editors. Scannin- Electron Microscopy. Physics of Image 585 I~'ormation and Microanalysis, 2"d Edition (Springer, 1998); Nie et al, Anal. Chem., 78:
I528-1534 (2006); Hecht et al, Journal Chemical Physics, 112: 7761-7774 (2000); Zhu et al, editors, Near-Field Optics: Principles and Applications (World Scientific Publishing, Singapore, 1999); Drmanac, International patent public-ation. WO20fl4t`0F
6683; Lehr et ale Anal. Chem.. 75: 2414-2420 (2003): Neuschafer et al, Biosensors &
Bioelectronics, 18: 489-590 497 (2003);'sieusebafer et aI, U.S. patent 6,289,144; and the like. Of particular interest is TIRFM. .-ir example, as disclosed by Neuschafer et al, U.S. patent E,289,144:
Lehr et al Ti10 20(-t '~.

1002791 In one aspect, instruments for use with arrays of the invention comprise three basic eomponents: (i) a flLiidics system for storing and transferring detection and processing 2595 reagents, e.g. probes, wash solutions, and the like, to an array; lii) a reaction chamber, or flow cell, holding or comprising an array and havint,: flow-through and temperature control capability; and (iii) an illumination and detection system. In one embodiment, a flow cell has a temperature control subsystem with ability to maintain temperature in the range from about 5-95`'C, or more specifically 10-85T, and can change temperature with a rate of about 2600 0.5-2 C per second.
100280] In one aspect, a tlow cell for 1" square 170 micrometer thick cover slips can be used which have been derivatized to bind macromolecular strLictures of the invention. " I`he cell encloses the "array" by sandwiching the glass and a gasket between two planes. One plane has an opening ol'sufficient size to permit imaging, and an indexing pocket for the 2605 cover slip. The other plane has an indexing pocket for the gasket, fluid ports, and a temperature control system. One fluid port is connected to a syringe pump which "pulls" or "Pusbes" fluid from the flow cell the other port is connected to a funnel like mixing chamber. The chamber, in turn is equipped with a liquid level sensor. The solutions are dispensed into the funnel, mixed if needed, then drawn into the flow cell.
When the level !6 10 sensor reads air in the funnels connection to the flow cell the pump is reversed a known amount to back the fluid up to the funnel. This prevents air from entering the flow cell. The cover slip surface may be sectioned off and divided into strips to accommodate tluid flow/capillary effects caused by sandwiching. Such substrate may be housed in an "open air" /"open face" chamber to promote even flow of the btifl'ers over the substrate by eliminating capillary flow effects, lmaging may be accomplished with a l00x objective using TIRF or epi illurnination and a 13 niega pixel 1-larnamatsu orca-cr-ag on a Zeiss axiovert 200, or like system. This configuration images RCR concatemers bound randomly to a substrate (non-ordered array). Imaging speed may be improved by decreasing the objective magnification power, using grid patterned arrays and increasing the number of 620 pixels of data collected in each image.
[00281] In one embodiment, four or more cameras may be used, preferably in the megapixel range. Multiple band pass filters and dichroic mirrors may also be used to collect pixel data across up to four or more emission spectra. To compensate for the lower light collecting power of the decreased magnification objective, the power of the excitation 525 light sourcz, can be increased. "1'hroug~put can be sncreased by ~Mwr ,nr ~v more flow cararn be . ;1 le s a.rÃ

being hy bridized/reacted. Because the probing of arrays can be non-setltaential, more than one imaging system can be used to collect data from a set of arrays, further decreasing assay timc.
2630 1002821 During the imaging process, it is preferable that the substrate remain in focus.
Some key factors in maintaining focus are the flatness of the substrate, orthogonality of the substrate to the focus plane, and mechanical forces on the substrate that may defonn it.
Substrate flatness can be well-controlled, and glass plates which have better than '/4 wave flatness are readily obtained. Uneven mechanical forces on the substrate can be minimized 21635 through proper design of the hybridization chamber. Orthogonality to the focus plane can be achieved by a -well adjusted, high precision stage. Auto focus routines generally take additional time to run, so it is desirable to run them oniy if necessary. In a preferred emboditnent. each imaLye is acquired and then analyzed usin.g a fast algorithm to determine if the image is in focus. If the image is out of f'ocus, the auto focus routine will be triggered.
?640 The system will then store the objectives Z position information to be used upon return to that section of that array during the next imaging cycle. By mapping the objective's Z
position at various locations on the substrate, it is possible to reduce the time required for substrate image acquisition.
1002831 In one aspect, suitable illumination and detection system for t7uorescence-based !645 signal is a Zeiss Axiovert 200 equipped with a TIRF slider coupled to an 80 milliwatt 532 nzn. solid state laser. The slider illuminates the substrate through the objective at the correct TIRF illumination angle. TIRF can also be accomplished without the use of the objective by illuminating the substrate though a prism optically coupled to the substrate.
Planar wave guides can also be used to implement TTRF on the substrate Epi illumination can also be 650 employed. "I'he liglat source can be rastered. spread beam, coherent, incoherent, and originate from.a single or multi-spectrum source.
(00284] One embodiment for the imaging system includes a 20x lens with a 1.25 nim field of view. A 10 megapixel carnera is used for detection. Such a system is able to image approximately 1.5 million concatemers attached to the patterned array at 1 micron pitch.
65 5 Under such a configuration, there are approximately 6.4 pixels per concatemer. The number of pixels per coneaterner can be adjusted by increasing or decreasing the field of view of the objective. For example, a 1 mm. field of view yields a value of 10 pixels per concatemer and a? mm field of view yields a value of 2.5 pixels per concatemer. The fleld of view may be a.dJusted relative to the mal;nif -,c ;;,, ~.nd numerical .~~ :->f the obgective to vield the 2660 lowest pixel count per concatemer that is still capable of being resolved by the optics. and in-iage analysis software.

[00285] Botb 'I'IRF and EPI illumination allow for almost any light source to be ttsed.
One illumination schema provides a cornmon set of monochromatic illumination sources (about 4 lasers for 6-8 colors) which is shared amongst imagers. Each ima~er collects data at 2665 a different wavelength at any given time and the light sources would be switched to the imagers via an optical switching system. In such an embodiment, the illtiimination source preferably produces at least 6, but more preferably 8 different wavelengths.
Such sources include gas lasers, multiple diode pumped solid state lasers combined through a fiber coupler, filtered Xenon Arc lamps, tunable lasers, or the more novel Spectralum Light 1-670 Engine, soon to be offered by Tidal Photonics. The Spectralum I,ight Engine uses prism to spectrally separate light. The spectrum is projected onto a Texas Instruments Digital Light Processor, which can selectively reflect any portion of the spectrum into a fiber or optical connector. This system is capable of monitoring and calibrating the power output across individual wavelengths to keep them constant so as to automatically compensate for ?675 intensity differences as bulbs age or between bulb changes. The following table represents examples of possible lasers, dyes and filters:

excitation laser filter emission filter Dye 407nin 405/12 436112 Alexa-405 4011421 407nm 405/12 546/10 cascade yel[ow 409/558 488nm 488/10 514/11 Alexa-488 492/517 543nin 546/10 540/565 Tamra 540/565 I3adipyr 543nni 546:`10 620/12 577/618 577/618 546i10 620/12 Alexa-594 594!613 635ntn 635/11 650/1 1 Alexa-635 632/647 635nm 635/11 Ate.xa700 702~723 f002861 In one aspect, imal;inl; is accomplished through a I00x objective.
"hbe excitation 680 light source is an 80 milliwatt diode pumped solid state laser. "I'Iiis light source has been used successfuliyr with "I'IRFM and EPI illumination techniques. The images are acquired using a 1.3 niegm pixel Ifamamatsu orca-er-ag camera and aZie-ss axiovert 200 inverted microscope. Tb.is configuration currently images DNBs bound randomly to a substrate at a 0.5 seconds exposure time.
2685 1002871 For handling multiple hybridization cycles a robotic station that is fully integrated with both the reaction chamber and detection svste:rri can be implemented for use with the present invention. Epifluorescence can be used for detecting greater than 10-20 fluorescent inolecules per target site. An advantage of using epiflnorescence is that it allows the use of probes of multiple colors with standard microscopes.
2690 1002881 ln one aspect, a two piece flow cell is used to house a I"
square, 170 pm thick cover slip, which has been derivatized and activated to bind DNBs. A side port is connected to a syringe pump that "pulls" or "push.es" fluid from the flow cell. A second port is connected to a funnel like mixing chaznber that is equipped with a liquid level sensor. The solutions are dispensed into the mixing chamber, aiixed if needed, then drawn into the flow 1695 cell. When the level sensor detects air in the funnel's connection to the t1ow cell, the pump is reversed a known amount to back the fluid up to the funnel. `I`bis prevents air from entering the flow cell. This chamber has worked well for cover slip sized slibstrates and may be used in modified form for the larger substrates. Such a three-axis robotic gantry pipetting system integrated with the hybridization chamber and imaging subsystem can be functionalized for ?700 fully automated probe pipettinlo.

Firlucials 1002891 In one embodiment, the regular pattern of capture cells is interrupted in such away.
as to encode location inforrnation into each acquired im.age. Approximately 1000 cells per image can be removed from the pattern to create a 10 bit code, which would represent up to ;705 1024 named locations on each substrate (Fig. 5).
1002901 The physical features of the coding region can be used as a reference to locate all pixels in the image du.rin- ima-e analvsis. while the code itself is used to verifi that the instrument imaged the correct area of the substrate. A key feature of the coding region is that each element is represented by a no-binding spots "empt~= area" block.
This eliminates 710 the need for fluorescent markers on the substrate. RCR products which are positive for a given probe-set define each r_lement's borders. 'I'his means that the region would still be recognizable even if only 5% to 10% of RCR products bound to the surface are positive for a given probe pool. In one embodiment, the code is readable if each coding element represents 50 capture cells M5 Kits vftlie invention [002911 In the commercialization of the methods described herein, certain kits for construction of random arrays of the invention and for using the same for various applications are particularly useful. Kits for applications of random arrays of the invention include, but are not limited to, kits for determining the nucleotide seqtitence of target ?720 polynucleotides. A kit typically coinprises at least one support having a surface and one or rrmore reagents u.ecessary or Liseful for constructin9 a random array of the iiivention or for carrying out an application therewith. Such reagents include, without li.mitation, nucleic acid primers, probes, adaptors. enzymes, and the like, and are each packaged in a container, such as, without limitation, a vial, tube or bottle, in a package suitable for commercial !725 distribution, such as, without limitation, a box, a sealed pouch, a blister pack and a carton.
The package typically contains a label or packaging insert indicating the uses of the packaged materials. As used herein, "packaginl; rnaterials" includes any article used in the packaging for distribution of reagents in a kit, including without limitation containers, vials, tubes, bottles, pouches, blister packaging, labels, tags, instruction sheets and package inserts.
!730 1002921 In another aspect the invention provides kits for sequencing a target polynucleotide comprising the followinti components: (i) a support having a planar surface having an array of optically resolvable discrete spaced apart regions, wherein each discrete spaced apart region has an area of less than 1pm'; (ii) a first set of probes for hybridizing to a plurality of concatemers randoinly disposed on the discrete spaced apart regions, the concatemers each ~735 containint, multiple copies of a DNA fragment of the target polynucleotide; and (iii) a second set of probes for hybridizing to the plurality of concatemers such that whenever a probe froi-n the first set hybridizes contiguously to a probe from the second set, the probes are ligated. Such kits may further include a ligase, a ligase buffer, and a hybridization buffer. In some embodiments, the discrete spaced apart regions may have capture 740 oligonucleotides attached and the concatemers may each have a region complementary to the capture oligonucleotides such that said concatemers are capable of being attached to the discrete spaced apart regions by formation of complexes between the capture oligonucleotides and the complementary regions of said concatemers.
[002931 In another aspect, the invention includes kits for circularizing DNA
fragments. In 745 an exemplary embodirrlc,nt, such a kit includes the components: (a) at least one adaptor oliaonucleotide for ligating to one or more DNA fragrnents and forming DNA
circles therewith (b) E r--m-a1 transf,r~se for attaÃ,hin x- a hornopoir:-mc- ta=1 to said DNA fragments to grov ide ad of s'l ' ligating a strand of said adaptor oligonucleotide to ends of said DNA fragment to form said 275o DNA circle, (d) a primer for annealing to a region of the strand of said adaptor olip-onucleotide, and (e) a DNA polymerase for extending the primer annealed to the strand in a rolling circle replication reaction. In a further embodiment, the above adaptor oligonucleotide may have a second end having a number of degenerate bases in the range of from 4 to 12. The above kit may further include reaction buffers for the terminal transferase, 2755 ligase, and DNA polymerase.
1002941 In still another aspect, the invention includes a kit fvr circularizin-DNA fragments using a CirciWigaserM enzyme (Epicentre Biotechnologies, Madison, WI), which kit comprises a volume exclusion polymer. In a further embodiment, the kit includes the following components: (a) reaction buffer for controlling pH and providing an optimized ?1760 salt composition for CircLigase, and (b) CircLigase cofactors. In another aspect, a reaction buffer for such kit comprises 0.5 M MOPS (pfl 7.5), 0.1 ivI KCI, 50 mM MgCi2, and 10 mM
DTT. In another aspect, such kit includes CircLigase, e.g. 10-I00 liL
Circl,igasc solution (at 100 unit/ L). Exemplary volume exclusion polymers are disclosed in U.S. patent 4,886,741, which is incorporated by reference, and include polyethylene glycol, polyvinylpyrrolidone, 1765 dextran sulfate, and like polymers. In one aspect, polyethylene glycol (PEG) is 50%
PEG4000. In one aspect, a kit for circle formation includes the following:
Ai-nount Camponent Final Conc.
2 la Circt.igaseTM I OX reaction buffer 0.5 ~Ia 1 inM ATP ?S ~eM

0.5 iL 50 mM 1\1nCl_ i? 5 rnVl 4 L 50% P1,.C;4000 ( 10%
1 2pL Circl,igaseTM ssDNA ligase (100 10 unitsl~LL [
units/ I,}
single stranded DNA template 0.5 1(1 rnol/ I_ sterile tivater Final reaction volume: 20 uL.
(00295] The above components can be used in a number of different protocols known in the 770 art, for example: (1) Heat DNA at 60- 96 C depending on the length of the DNA (ssDNA
templates that have a 5'- phosphate and a 3'-hydroxyl group); (2) Preheat 2.2X
reaction mix at 60`'`C for about 5-10 niin; (3} If DNA was preheated to 96'C cool it down at 60"C.Mix DNA and buffer at 60"C without cooling it down and incubate for 2-3h; (4) Heat-inactivate enzyme to stop the ligation reaction.
1002961 present invention ma.~~ be better Lindersto~.~d by reference to the folfoeving non-~: .. . u fthe in4 .

examples are presented in order to more fully illustrate preferred embodiments of the invention, but should in no Lvay be construed as limiting the broad scope of the invention.
EXAMPLES
2780 Example 1: RCR ba.sed formation and attachment of DNBs 1002971 Two synthetic targets were co-amplified. About one million molecules were captured on the glass surface, and then probed for one of the targets. After imaging and photo-bleaching the first proben the second target was probed. Successive hybridization with amplicon specific probes sliowed that each spot on the array corresponded uniquely to either 2785 one of the two amplicon sequences. It was also confirmed that the, probe could be removed through heating to 70"C and then. re-hybridized to produce equally strong signals.
Example 2: Validation of circle formation and amplification [002981 The circle formation and amplification process was validated using E.
coli DNA
(Fig. 6). A universal adaptpr, v,jhich also served as the binding site for capture probes and 2790 RCR primer, was ligated to the 5` end of the target molecule using a universal template DNA
containing degenerate bases for binding to all genomic sequences. The 3" end of the target molecule was modified by addition o:f a poly-dA tail using terminal transferase. The modified target was then circularized using a bridging template complementary to the adaptor and to the oligo-dA. tail.

?795 Example 3: Validation of li~ation with cor~densed concatenr~ers [00299] The ability for probe ligation to occur with the condensed concatemers was tested.
Reactions were carried out at ?0 C for 10 min crsing ligase, followed by a brief wash of the chamber to remove excess probes. The ligation of a 6-mer and a labeled 5-iner produced signal levels comparable to that of an 1.1-mer. Software modules, including, image analysis goo of random arrays, were tested on simulated data for whole genome sequence reconstruction.
Example 4: Identific-ation oftargets from muiti le athogcns ~~le arrav 1003001 PCR products from diagnostic regions of Bacillus anthracis and Yersinia pestis were converted into single stranded DNA and attached to a universal adaptor.
'I`hese "
samples were then mixed and replicated together usinLy RCR and deposited onto the chip 805 surface as a random array. Successive hybridiration with anxplicon specific probes showed that each spot on the array corresponded wiiquely to either om~ of the two arn.plicon il i?c . . _ . t ~ "bes { F'i g. 7 ), . . . ., demonstrating sensitivitlv and specificity for identifying DNA present in submicron size DNA nano-balls having about 100-1000 copies of a DNA fragment generated by the RCR
281 ci reaction.

1003011 A 1 55 bp amplicon sequence froni B. anthracis and a 275 bp amplicon sequence from Y. pestis were amplified using standard PCR techniques with PCR primers in which one primer of the pair was phosphorylated. A single stranded form of the PCR
products was generated by degradation of the phosphorylated strand using lambda exonuclease. The 5' 2815 end of the remaining strand was then phosphorylated using 44 DNA
polynucleotide kinase to allow li~atior~ of the single stranded product to the universal adaptor.
The universal adaptor was ligated using T4 DNA ligase to the 5' end of the target molecule, assisted by a template oligonucleotide compleincntary to the 5' end of the targets and 3' end of the universal adaptor. The adaptor ligated targets were then circularized using bridging 2820 oligonucleotides with bases complementary to the adaptor and to the 3' end of the targets.
Linear DNA molecules were removed by treating with exoaiuclease I. RCR were generated by mixing the single-stranded samples and usiiig Phi29 polymerase to replicate around the circularized adaptor-target molecules with the bridging oligonucleotides as tlle initiating primers. The RCR products were captured on the glass slide via the capture oligonucleotide, ?825 which was attached to derivatized glass coverslips and was complementary to the universal adaptor sequence.

[003021 Arrayed target nano-ball molecules derived from B. anthracis and Y.
pestis PCR
amplicons were probed sequentially witb. TAMRA-labeled 11-rrzer probes cornplementary to the universal adaptor sequence, or 11-iner probes complementary to one of the two amplicon 830 sequences By overlaying the irnages obtained from successive hybridization of 3 probes, O`ig. 7) it can be seen that most of the arrayed molecules that hybridized with the adaptor probe (blue spots) would onlv hybridize to either the amplicon I probe (red spots) or the amplicon 2 probe (green spots), with veryr few that would hybridize to bot11, This specific hvbridiza.tion pattern demonstrated that each spot on the array contained only oiie type of 835 sequence, either the B anthracis amplicon or the Y, pestis ampiicon. It also demonstrated that the rSBH process was able to distinguish target molecules of different sequences deposited onto the arrav by using sequence specific probes.

Example 5: Decodin ~ base :sitioz~ in arra~ed DNBs created from ~0 r~er oliwnucleotide with dezeqerate bases 2840 1003031 Indir:idual molecules of a synthetic oligonucleotide containing a degenerate base were divided into 4 sub-popolatians. each haviiig either an A, C, G or T base at that particular position. An array ofDMs created from this synthetic DNA can have about 25%
of spots with each of the bases. Four successive hybridization and ligation of pairs of probes specific to each of the 4 bases identified the stib-populations (Fig. 8).
2845 1003041 A S' phosphorylated, 3` TAMRA-labeled pentamer oligonucleotide was paired with one of the four hexamer oligonucleotides. Each of these 41igation probe pairs hybridize to either an A, C, G or T-containing version of the target. Discrimination scores of greater than 3 were obtained for most targets, demonstrating the ability to identify single base differences between the nanoball targets. `].'he discrimination score is the highest spot score divided by.
28-io the average of the other 3 base-specific signals of the same spot.
Adjusting the assay conditions (buffer composition, concentrations of all compoiients, time and temperature of each step in the cycle) can result in higher signal to background allowing for calculation of full match to mismatch ratios.
1003051 A similar ligation assay was performed on the spotted arrays of 6-mer probes, In !855 this case full-match/background ratio was about 50 and the average full match/mismatch ratio was 30. The results further demonstrated the ability to determine partial or complete sequences of DNA present in DNBs by increasing the number of consecutive probe cycles or by using 4 or more probes labeled with different dyes per each cycle.
[00306] To identify the sub-populations, a set of 41igation probes specific to each of the 4 860 bases was used. A 5'piZosphorylated, 3' TAMRA-labeled pentamer oligonucleotide corresponding to position 33-37 ofrl`lA with sequence CAAAC (probe TIA9b) was paired with one oI'the following hexamer oligonucleotides corresponding to position 27-32 :
ACTGTA (probe T 1 A9a), ACTGTC (probe I1 A l 0a), AC'I`GTG (probe T 1 A l 1 a), AC."I'GT"I' (probe Ti A 12a). Each of these 4 ligation probe pairs should bybridizc to either an A, C. G
865 or'T' containing version of'1`lA. For each bybridization cycle, the probes were incubated with. the array in a ligation/bybridization buf'fer containing T4 DNA ligase at 2WC. for 5 minutes. Excess probes ~Nrere washed off at 2C} C and images were taken with the TIRF
microscope. Bound probes were stripped to prepare for the next round of bybridization.
1003071 An adaptor specific probe (Brprb3) was hybridized to the array to establish the 37V IIN `lÃ.1131d f I-. at V.4 ;6~ ¾ .._... heSE
k t" , hybridized successively to the array: the spots hybridized to the A-specific ligation probe pair are shown as red in figure 5, the C-specific spots are green, G-specitic spots are yellow and the "I,-specilic spots are cyan. In figure 3, circle A indicates the position of one of the spots hybridized to both the adaptor probe and the A-specific ligation probe pair, suggesting 2$75 that the DNA ar-rayed at this spot is derived from a molecule of "I'1A
that contains an A at position 32. It is clear that most of the spots associated with only one, of the 4 ligation probe pairs, allowing identification of the base at position 32 to be determined specifically.
1003081 Using an in-house image analysis program, spots were identified using the images taken for the hybridization cycle using the adaptor probe. The same spots were also 28so identified, and the fluorescent signals were quantified for subsequent cycles, with the base-specif c ligation probes. A discrimination score was calculated for each signal for each base-specific signal of each spot. The discrimination score is the spot score divided by the average of the other 3 base-specific signals of the same spot. For each spot, the highest of the 4 base-specil~ic discrimination scores was compared with the second highest score. If the ?885 ratio of the two was above 1.8, then the base corresponding to the maximum discrimination score was selected for the base calling. In thls analysis over 500 spots were successf'ully base-called and the average discrirnination score was 3.34. The average full niatch signal was 272, while the average single mismatch signal (signals from the un-selccted bases) was 83.2. Thus the full match / mismatch ratio was 3.27. The image background noise was !890 calculated by quantifying signals from randomly selected empty spots and the average si~r~al of these empty spots was 82.9. Thus the full match I background noise ratio was 3.28. In these experiments the mismatch discrimination was limited by the low full match signal relative to the background.

Example 6: Decodin 2 degenerate bases at the en.d of a synthetic 80-mer 895 oEi~aonucleotide using aprobe_-anchor ligation. assa~r 100309J A synthetic oligonucleotide containina 8 d.ecienerate bases at the S' end was used to simtilate random genomic DNA ends. The DNA-nanoballs created from this oli-onucleotide will have these 8 degenerate bases placed directly next to the adaptor sequence. `1-o demonstrate the feasibility of sequencing the 2 unkno,~vn bases adjacent to the known 400 adaptor sequence using a probe-anchor ligation approach, a 12-nler oligonucleotide with a specific sequence to hybridize to the 3' end of the adaptor sequence vv~as used as the anchor, and a set of 16 TAN1RA-labeled oligonucleotides in the f'orm of I3BNivNN'v'V
were used as [00310] Using a subset of the BBNNNNNN probe set (namely GA, GC, GG and G`I'in the 2905 place of BB), spots could be identified on the nano-ball array created from targets that specifically bind to one of these 4 probes, with an average full mateh/r~i isinatch ratio of over 20 (Fig,. 9), Example 7: F'roducin z stractarecl nano-ball arrays [003111 Ordered array lines of capture probe separated on averagle by 5 um were prepared.
2910 Lines were produced by using a pulled glass capillary beveled at 45 decyrc,cs to a tip size of 5 pm, loaded with 1 1 of 5 iVl capture probe in water, and drawn across the glass slide by a precision gantry robot. DNBs were allowed to attach to the surface of the coverslip and then detected with a probe specific for the adaptor. Fig. 10 shows the high density attachment to regions where a capture probe was deposited on the surface, indicating that DNBs can be 2915 arranged in a grid if a substrate with submicron binding sites is prepared.

Example 8: Demonstrating circle formation with multiple adaptors [00312] A synthetic target DNA of 70 bases in length and a PCR derived fragment of' 200-300 bp in length was obtained from a double stranded product by phosphorylation of one of the primers and treatment with lambda exonuclease to reinove the phosphorylated strand.
?920 The single stranded fi:aginent was ligated to an adaptor for circularization. Polymerization.
type IIs restri.etion enzyme digestion and re-ligation with a new adaptor was performed as described herein.

100313J Demonstration that the process was successful was accomplished using RCR
amplification of the final derived circles. Brzefly, the DNA circles were incubated with !925 primer complementary to the last introduced adaptor and Phi29 polymerase for I hour at 30 C to generate a single concatemer molecule consisting of hundreds of repeated copies of the original DNA circle. Attachment of the RCR products to the surface of coverslips could also be accomplished by utilizing an adaptor sequence in the coneatemer that is complementary to an attached oligonucleotide on the surface. Hybridization of adaptor 930 unique probes was used to demonstrate that the individual adaptors were incorporated into the circle and ultiirrately the R.CR product. "f'o demonstrate that the adaptors were incorporated at the expected positions within the circle, sequence specific probes (labeled 5-mers) were used for the svnthetic or PCR derived scqrience such that ligation may occur to an unlabeled anchor probe that recognizes the terminal sequence of thc adaptor. Cloning and 7.3~ ..:l~~d to vL;~'If~' D*4 ~1f' pr4}'L'6' generating clean ssDNA after each circle cutting which allowed the use of the same circle closing chemistry for eacli of the adaptor incorporations.

Claims (42)

1. A method of determining the identification of a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:
(a) providing a plurality of concatemers, wherein each concatemer comprises a plurality of monomers and each monomer comprises:
i) a first target domain of'said target sequence comprising a first set of target detection positions;
ii) a first adaptor comprising a Type IIs endonuclease restriction site;
iii) a second target domain of said target sequence comprising a second set of target detection positions; and iv) a second interspersed adaptor comprising a Type IIs endonuelease restriction site;
(b) identifying said first nucleotide.

2. A method according to claim 1 wherein said target sequence concatemers are immobilized on a surface.

3. A method according to claim 2 wherein said identifying step comprises:
(a) contacting said concatemers with a set of sequencing probes each comprising:
i) a first domain complementary to one of said adaptors;
ii) a unique nucleotide at a first interrogation position; and iii) a label;
under conditions wherein if said unique nucleotide is complementary to said first nucleotide, a sequencing probe hybridizes to said concatemer; and (b) identifying said first nucleotide.

4. A method according to claim 2 wherein each adaptor comprises an anchor probe hybridization site, and said identifying step comprises:
(a) hybridizing anchor probes to said anchor probe hybridization sites;
(b) hybridizing sequencing probes to target detection positions adjacent to said adaptors;
(c) ligating adjacent hybridized sequencing and anchor probes to form ligated probes; and (d) detecting said ligated probes to identify said first nucleotide.

5. A method according to claim 2 wherein each adaptor comprises an anchor probe hybridization site, and said identifying step comprises:
(a) hybridizing anchor probes to said anchor probe hybridization sites;
(b) adding a polymerase and at least one dNTP comprising a label, under conditions whereby if said dNTP is perfectly complementary to a detection position, said dNTP is added to the anchor probe to form an extended probe, thereby creating an interrogation position of the extended probe; and (c) determining the nucleotide at the interrogation position of the extended probe.

6. A method according to claim 2 wherein a nucleotide at a second detection position is identified.

7. A method according to claim 6 wherein a nucleotide at a third detection position is identified.

8. A method according to claim 7 wherein a nucleotide at a fourth detection position is identified.

9. A method according to claim 8 wherein a nucleotide at a fourth detection position is identified.

10. A method according to claim 9 wherein a nucleotide at a sixth detection position is identified.

11. A method according to claim 2 wherein said surface is functionalized,

12. A method according to claim 11 wherein said functionalized surface comprises functional moieties selected from the group consisting of amines, silanes, and hydroxyls.

13. A method according to claim 2 wherein said surface comprises a plurality of spatially distinct regions comprising said immobilized concatemers.

14. A method according to claim 2 wherein said concatemers are immobilized on said surface using capture probes.

15. A method according to claim 1 further comprising fragmenting genomic nucleic acid to form target sequences.

16. A method according to claim 1 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are the same.

17. A method according to claim 1 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are different.

18. A substrate comprising a plurality of immobilized concatemers, each monomer of said concatemer comprising:
a) a first target sequence;
b) a first adaptor comprising a Type IIs endonuclease restriction site;
c) a second target sequence; and d) a second interspersed adaptor comprising a Type IIs endonuclease restriction site.

19. A substrate according to claim 18 wherein each monomer further comprises a third target sequence and a third interspersed adaptor comprising a Type IIs endonuclease restriction site.

20. A substrate according to claim 19 each monomer further comprises a fourth target sequence and a fourth interspersed adaptor comprising a Type IIs endonuclease restriction site.

21. A substrate according to claim 18 wherein said substrate is glass.

22. A substrate according to claim 21 wherein said glass is functionalized.

23. A substrate according to claim 18 wherein said substrate comprises capture probes and said concatemers are immobilized by hybridization to said capture probes.

24. A substrate according to claim 18 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are the same.

25. A substrate according to claim 18 wherein the Type IIs endonuclease restriction sites of said first and second adaptors are different.

26. A substrate according to claim 18 wherein said target sequences are genomic nucleic acid sequences.

27. A substrate according to claim 26 wherein said genomic nucleic acid sequences are human.

28. A method of inserting multiple adaptors in a target sequence comprising:
(a) ligating a first adaptor to one terminus of said target sequence, wherein the adaptor comprises a binding site for a restriction enzyme;
(h) circularizing the product from step (i) to create a first circular polynucleotide;
(c) cleaving the circular polynucleotide with a restriction enzyme, wherein the restriction enzyme is able to bind to the binding site within the first adaptor;
(d) ligating a second adaptor, wherein said second adaptor comprises a binding site for a restriction enzyme;
(e) circularizing the product from step (d) to create a second circular polysnucleotide;
wherein steps (c) through (e) are optionally repeated to insert a desired number of adaptors in the target sequence.

29. A method according to claim 28 wherein said binding site of said first adaptor comprises a Type IIs endonuclease restriction site.

30. A method according to claim 28 wherein said binding site of said second adaptor comprises a Type IIs endonuclease restriction site.

31. A method according to claim 28 wherein said circularization step comprises adding a CircLigase.TM. enzyme.

32. A method according to claim 28 wherein said circularization step comprises:
(a) adding a circularization sequence to a second terminus of said target sequence;
(b) hybridizing a bridge template to at least a portion of said adaptor and a portion of said circularization sequence;
(c) ligating said first and second termini together to circularize the target sequence.

33. A method for identifying a nucleotide sequence of a target sequence, the method comprising the steps of:
(a) providing a plurality of interspersed adaptors within a target sequence, each interspersed adaptor having at least one boundary with the target sequence; and (b) determining the identity of at least one nucleotide adjacent to at least one boundary of at least two interspersed adaptors, thereby identifying a nucleotide sequence of the target sequence.

34. A library of polynucleotides comprising more than one nucleic acid fragment, each fragment comprising a plurality of interspersed adaptor, wherein each interspersed adaptor has t least one end having different non-cross-hybridizable sequence with respect to the sequences of every other interspersed adaptor of the plurality.

35. The library of claim 34, wherein the plurality of interspersed adaptors is in a predetermined order.

36. The library of claim 35, wherein the predetermined order of the interspersed adaptors is the same for every nucleic acid fragment.

37. The library of claim 34, wherein each of said nucleic acid fragments is a closed single stranded DNA circle.

38. A method of identifying a nucleotide sequence of a target sequence, the method comprising the steps of:
(a) providing an amplicon from each of a plurality of fragments of the target sequence, each fragment containing a plurality of interspersed adaptors at predetermined sites, and each amplicon comprising multiple copies of a fragment and the amplicons including a number of fragments that substantially covers the target sequence;
(b) providing a random array of amplicons fixed to a surface at a density such that at least a majority of the amplicons are optically resolvable;
(c) hybridizing one or more sequencing probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more sequencing probes and complementary sequences on the interspersed adaptors, (d) identifying at least one nucleotide adjacent to at least one interspersed adaptor by extending the one or more sequencing probes in a sequence specific reaction;
and (e) repeating steps (e) and (d) until a nucleotide sequence of the target sequence is identified.

39. A method of identifying a nucleotide sequence of a target sequence, the method comprising the steps of:
(a) providing a random array of concatemers fixed to a planar surface, wherein said surface has an array of optically resolvable discrete spaced apart regions, and wherein each discrete spaced apart region has an area of less than 1 µm2 and substantially all such regions have at most one of said concatemers attached, each concatemer comprising multiple copies of a fragment of the target sequence, each such fragment continuing a plurality of interspersed adaptors at predetermined sites, and the number of different concatemers such that their respective fragments substantially cover the target polynucleotide;
(b) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on the concatemers;
(c) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the one or more probes and complementary sequences on the concatemers;
(d) ligating probes from the first and second sets which are hybridized to a concatemer at contiguous sites;
(e) identifying the sequences of the ligated probes; and (f) repeating steps (b) through (e) to identify the nucleotide sequence of the target sequence.

40. A method of identifying a nucleotide sequence of a target sequence, the method comprising the steps of:
(a) providing a plurality of concatemers from the target sequence, each concatemer comprising multiple copies of a fragment of the target sequence, each fragment containing a plurality of interspersed adaptors at predetermined sites;
(b) providing a random array of concatemers fixed to a surface at a density such that at least a majority of the concatemers are optically resolvable;
(c) identifying a sequence of at least a portion of each fragment adjacent to at least one interspersed adaptor in at least one concatemer, thereby identifying a nucleotide sequence of the target sequence.

41. The method of claim 40, wherein said plurality of concatemers includes a number of fragments such that said fragments substantially cover said target sequence.

42. The method of claim 41 further comprising a step of reconstructing a nucleotide sequence of the target sequence from the identities of the sequences of said portions of said fragments of said concatemers.