Methods and compositions for long fragment read sequencing
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12P-019/34
출원번호
US-0816365
(2010-06-15)
등록번호
US-8592150
(2013-11-26)
발명자
/ 주소
Drmanac, Radoje
Peters, Brock A.
Alexeev, Andrei
Hong, Peter
출원인 / 주소
Complete Genomics, Inc.
대리인 / 주소
Kilpatrick Townsend & Stockton LLP
인용정보
피인용 횟수 :
34인용 특허 :
92
초록▼
The present invention is directed to methods and compositions for long fragment read sequencing. The present invention encompasses methods and compositions for preparing long fragments of genomic DNA, for processing genomic DNA for long fragment read sequencing methods, as well as software and algor
The present invention is directed to methods and compositions for long fragment read sequencing. The present invention encompasses methods and compositions for preparing long fragments of genomic DNA, for processing genomic DNA for long fragment read sequencing methods, as well as software and algorithms for processing and analyzing sequence data.
대표청구항▼
1. A method of obtaining sequence information from a genome, said method comprising: (a) providing a population of first fragments of said genome;(b) preparing emulsion droplets of said first fragments, such that each emulsion droplet comprises a subset of said population of first fragments;(c) frag
1. A method of obtaining sequence information from a genome, said method comprising: (a) providing a population of first fragments of said genome;(b) preparing emulsion droplets of said first fragments, such that each emulsion droplet comprises a subset of said population of first fragments;(c) fragmenting said first fragments, thereby obtaining a population of second fragments within each emulsion droplet, such that said second fragments are shorter than said first fragments;(d) combining individual emulsion droplets comprising said second fragments with individual emulsion droplets comprising adaptor tags or adaptor tag combinations, thereby forming fused droplets;(e) ligating said second fragments with said adaptor tags or adaptor tag combinations within the fused droplets to form tagged fragments;(f) combining the fused droplets to produce a mixture containing tagged fragments;(g) obtaining sequence reads from tagged fragments in the mixture;(h) assembling the sequence reads to produce assembled sequence information for the genome, wherein the assembled sequence information comprises heterozygous loci; and(i) phasing the heterozygous loci using sequence information from the adaptor tags. 2. The method of claim 1, wherein said emulsion droplets of said adaptor tags each individually comprises at least two sets of different tag components such that fragments in at least some of said emulsion droplets are tagged with different combinations of said tag components in said ligating step (e). 3. The method of claim 2, wherein at least 1000 of said emulsion droplets in said ligating step (e) comprise fragments tagged with different combinations of said tag components. 4. The method of claim 3, wherein at least 10,000, 30,000, or 100,000 of said emulsion droplets in said ligating step (e) comprise fragments tagged with different adaptor tags or adaptor tag combinations. 5. The method of claim 2, wherein said tag components are from a set of over 1000 distinct barcodes prepared as a population of liquid drops in oil. 6. The method of claim 1, wherein said emulsion droplets of said first fragments comprise only 1-5 first fragments in each droplet. 7. The method of claim 1, wherein said emulsion droplets of said second fragments or said emulsion droplets of said adaptors further comprise ligase. 8. The method of claim 1, wherein each adaptor tag or adaptor tag combination comprises a segment that is common to all adaptor tags and a segment comprising an identifier sequence. 9. The method of claim 1, wherein each adaptor tag is blocked at one end to control orientation of the tag when ligated to a genome fragment. 10. The method of claim 1, wherein step (c) comprises amplifying the first fragments so as to replace nucleotides therein with the nucleotide analogs, and then either excising the nucleotide analogs or producing a nick either immediately 3′ or 5′ to the nucleotide analogs, thereby forming gapped nucleic acids. 11. The method of claim 1, wherein the adaptor tag or adaptor tag combination comprises eight bases or a combination of 2×5 bases. 12. The method of claim 1, wherein the sequence reads in step (g) are obtained by a process comprising probe anchor ligation. 13. A method of obtaining sequence information from a genome, said method comprising: (a) providing a population of first fragments of said genome;(b) preparing emulsion droplets of said first fragments, such that each emulsion droplet comprises a subset of said population of first fragments;(c) fragmenting said first fragments, thereby obtaining a population of second fragments within each emulsion droplet, such that said second fragments are shorter than said first fragments;(d) combining individual emulsion droplets comprising said second fragments with individual emulsion droplets comprising adaptor tags or adaptor tag combinations, thereby forming fused droplets;(e) ligating said second fragments with said adaptor tags or adaptor tag combinations within the fused droplets to form tagged fragments;(f) combining the fused droplets to produce a mixture containing tagged fragments;(g) obtaining sequence reads from tagged fragments in the mixture,(h) combining sequence reads from tagged fragments having the same adaptor tags to produce sequences of longer contiguous regions; and(i) assembling the sequence reads into sequence information for the genome wherein the sequence information comprises said sequences of longer contiguous regions. 14. The method of claim 13, wherein said emulsion droplets of said adaptor tags each individually comprises at least two sets of different tag components such that fragments in at least some of said emulsion droplets are tagged with different combinations of said tag components in said ligating step (e). 15. The method of claim 13, wherein said emulsion droplets of said second fragments or said emulsion droplets of said adaptors further comprise ligase. 16. The method of claim 13, wherein each adaptor tag or adaptor tag combination comprises a segment that is common to all adaptor tags and a segment comprising an identifier sequence. 17. The method of claim 13, wherein each adaptor tag is blocked at one end to control orientation of the tag when ligated to a genome fragment. 18. The method of claim 13, wherein step (c) comprises amplifying the first fragments so as to replace nucleotides therein with the nucleotide analogs, and then either excising the nucleotide analogs or producing a nick either immediately 3′ or 5′ to the nucleotide analogs, thereby forming gapped nucleic acids. 19. The method of claim 13, wherein the adaptor tag or adaptor tag combination comprises eight bases or a combination of 2×5 bases. 20. The method of claim 13, wherein the sequence reads in step (g) are obtained by a process comprising probe anchor ligation. 21. The method of claim 13, wherein step (h) generates sequence data equivalent to sequencing single DNA molecules of greater than 100 kb. 22. The method of claim 13, which is a method for sequencing a complete diploid genome. 23. The method of claim 13, which is a method for haplotyping a diploid chromosome.
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