Two-adaptor library for high-throughput sequencing on DNA arrays
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12N-015/66
C12N-015/64
출원번호
US-0981605
(2007-10-31)
등록번호
US-9476054
(2016-10-25)
발명자
/ 주소
Drmanac, Radoje T.
Callow, Matthew
Drmanac, Snezana
출원인 / 주소
Complete Genomics, Inc.
대리인 / 주소
Kilpatrick Townsend & Stockton LLP
인용정보
피인용 횟수 :
1인용 특허 :
140
초록▼
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 inventi
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.
대표청구항▼
1. An array configured for identifying nucleotide sequences of a target nucleic acid, comprising a plurality of amplicons randomly disposed on a surface in a pattern, wherein each of the amplicons comprise multiple copies of a particular monomer,wherein the particular monomer in each amplicon compri
1. An array configured for identifying nucleotide sequences of a target nucleic acid, comprising a plurality of amplicons randomly disposed on a surface in a pattern, wherein each of the amplicons comprise multiple copies of a particular monomer,wherein the particular monomer in each amplicon comprises a first adaptor, a second adaptor, and subfragments of the target nucleic acid, and has been formed by a process comprising: (i) forming a first circular DNA comprising a fragment of said target nucleic acid and a first adaptor, wherein the first adaptor comprises a binding site for a restriction enzyme that cleaves DNA at a cleavage site separated from said binding site by at least six nucleotides;(ii) forming a linearized DNA by a process that comprises cleaving the first circular DNA at a site that is internal to the fragment of the target nucleic acid using a restriction enzyme that binds to said binding site on the first adaptor, thereby producing a first subfragment and a second subfragment of the target nucleic acid separated by the first adaptor; and(iii) inserting a second adaptor so as to form a second circular DNA comprising in order: the first subfragment, the first adaptor, the second subfragment, and the second adaptor;wherein the second adaptor is different from the first adaptor and comprises two probe hybridization sites, one adjacent or close to the 3′ end of the adaptor and one adjacent or close to the 5′ end of the adaptor such that the adaptor is configured for determining target sequences in the monomer both upstream and downstream from the adaptor. 2. The array of claim 1, wherein each amplicon of the array comprises a different fragment of the target nucleic acid. 3. The array of claim 1, wherein the target nucleic acid is genomic DNA. 4. The array of claim 1, wherein the surface comprises more than 100,000 amplicons per square millimeter. 5. The array of claim 4, wherein at least 70% of said amplicons are optically resolvable. 6. The array of claim 1, wherein the amplicons are bound to discrete spaced-apart regions on the surface. 7. The array of claim 6, wherein the amplicons are randomly disposed amongst said discrete spaced apart regions. 8. The array of claim 6, wherein each discrete spaced apart region has an area of less than 1 μm2. 9. The array of claim 6, wherein the amplicons are bound to the discrete spaced-apart regions non-covalently. 10. The array of claim 6, wherein the area of each discrete region corresponds to the size of a single amplicons. 11. A combination configured for identifying nucleotide sequences of a target nucleic acid, comprising: (a) an array according to claim 1; and(b) nucleic acid probes configured to hybridize to at least one of said probe hybridization sites. 12. The combination of claim 11, wherein said nucleic acid probes are anchor probes, and the combination further comprises: (c) sequencing probes configured to hybridize to a target sequence adjacent to the anchor probe that is hybridized to the probe hybridization site, and thereafter to be ligated to said anchor probe. 13. A method for identifying at least one nucleotide of a target nucleic acid, comprising: (a) providing an array configured for identifying nucleotide sequences of a target nucleic acid, the array comprising a plurality of amplicons having been formed and randomly disposed on a surface in accordance with claim 1; and(b) identifying at least one nucleotide of the target nucleic acid adjacent or close to the second adaptor. 14. The method of claim 13, comprising identifying at least one nucleotide of the target nucleic acid adjacent or close to the 3′ end of the second adaptor, and identifying at least one nucleotide of the target nucleic acid adjacent or close to the 5′ end of the second adaptor. 15. The method of claim 13, wherein step (b) comprises forming a hybridization product between the monomer and a nucleic acid, whereby the nucleic acid comprises an anchor sequence hybridized to a hybridization site adjacent or close to the 3′ or 5′ end of the second adaptor, and at least one informative base hybridized to the target nucleic acid upstream or downstream from said hybridization site. 16. The method of claim 15, wherein the nucleic acid is formed by hybridizing an anchor probe to the hybridization site on the second adaptor, hybridizing a sequencing probe comprising the informative base adjacent to the anchor probe, and then ligating the sequencing probe to the anchor probe. 17. The method of claim 13, wherein step (b) comprises: (i) contacting the plurality of amplicons with anchor probes under conditions where the anchor probes bind to a hybridization site adjacent or close to the 3′ or the 5′ end of the second adaptor;(ii) contacting the plurality of amplicons with sequencing probes having at least one informative base under conditions wherein the sequencing probes hybridize to the target nucleic acid adjacent to the anchor probe when the sequencing probe is substantially complementary to the target nucleic acid;(iii) ligating anchor probes and sequencing probes hybridized to adjacent positions on the amplicons, thereby forming ligation products; and(iv) detecting the ligation products. 18. The method of claim 13, wherein step (b) comprises: (i) contacting the plurality of amplicons with first anchor probes under conditions where the first anchor probes bind to a first hybridization site adjacent or close to the 3′ end of the second adaptor;(ii) contacting the plurality of amplicons with second anchor probes under conditions where the second anchor probes bind to a second hybridization site adjacent or close to the 5′ end of the second adaptor;(iii) contacting the plurality of amplicons with sequencing probes having at least one informative base under conditions wherein the sequencing probes hybridize to the target nucleic acid adjacent to the first or second anchor probe when the sequencing probe is substantially complementary to the target nucleic acid;(iv) ligating anchor probes and sequencing probes hybridized to adjacent positions on the amplicons, thereby forming ligation products; and(v) detecting the ligation products.
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