Methods for obtaining a sequence

Patent metadata

Field	Value
Publication number	US-9725765-B2
Application number	US-201213608770-A
Country	US
Kind code	B2
Filing date	Sep 10, 2012
Priority date	Sep 9, 2011
Publication date	Aug 8, 2017
Grant date	Aug 8, 2017

Abstract

The invention generally relates to methods for obtaining a sequence, such as a consensus sequence or a haplotype sequence. In certain embodiments, methods of the invention involve determining an amount of amplifiable nucleic acid present in a sample, partitioning the nucleic acid based upon results of the determining step such that each partitioned portion includes, on average, a subset of unique sequences, sequencing the nucleic acid to obtain sequence reads, and assembling a consensus sequence from the reads.

First claim

What is claimed is:

1. A method for analyzing a plurality of target polynucleotides, the method comprising:
(a) providing a sample comprising a plurality of target polynucleotide fragments, wherein each target polynucleotide fragment has a length of 5 kb to 40 kb;
(b) performing a size selection on the plurality of target polynucleotide fragments to select 5-7 kb fragments;
(c) ligating amplification adaptors at each end of at least a portion of the 5-7 kb fragments to produce a library of amplifiable fragments, wherein the adaptors serve as primer binding sites;
(d) quantitating via qPCR to establish a number of amplifiable fragments in the library of amplifiable fragments;
(e) determining a dilution factor based on the number of amplifiable fragments in the library of amplifiable fragments;
(f) diluting and partitioning the library into a plurality of partitions based on the dilution factor such that each partition comprises, on average, a unique subset of 500 or more of the amplifiable fragments;
(g) amplifying via polymerase chain reaction the partitioned amplifiable fragments;
(h) subjecting the partitions to polynucleotide fragmentation thereby generating a set of smaller fragments; and
(i) obtaining partial or complete sequence information from at least a subset of the smaller fragments.

2. A method for analyzing a plurality of target polynucleotides, the method comprising:
(a) providing at least one sample comprising a plurality of target polynucleotide fragments, wherein each target polynucleotide fragment has of a length of 5 kb to 40 kb;
(b) performing a size selection on the plurality of target polynucleotide fragments to select 5-7 kb fragments;
(c) ligating amplification adaptors to each end of at least a portion of the 5-7 kb fragments to produce a library of amplifiable fragments, wherein the adaptor serves as a primer binding site;
(d) quantitating via qPCR to establish a number of amplifiable fragments in the library of amplifiable fragments;
(e) determining a dilution factor based on the number of amplifiable fragments in the library of amplifiable fragments;
(f) diluting and partitioning the library based on the dilution factor into a plurality of partitions such that each partition comprises, on average, a unique subset of 500 or more of the amplifiable fragments;
(g) amplifying via a polymerase chain reaction the partitioned amplifiable fragments;
(h) subjecting the partitions to polynucleotide fragmentation thereby generating a set of smaller fragments;
(i) coupling a partition specific barcode to at least a subset of the smaller fragments; and
(j) obtaining partial or complete sequence information from at least a subset of the smaller fragments coupled with the partition specific barcodes.

3. The method of claim 1 , wherein the adaptors coupled to each end of the 5-7 kb fragments comprise a first adaptor and a second adaptor, and the first adaptor is not the same as the second adaptor.

4. The method of claim 1 , wherein the smaller fragments within a partition are labeled with one or more partition specific barcodes.

5. The method of claim 4 , wherein the barcodes are nucleic acid sequences.

6. The method of claim 4 , wherein the barcodes are greater than 3 nucleotides long.

7. The method of claim 4 , wherein the barcodes are greater than 5 nucleotides long.

8. The method of claim 4 , wherein the one or more partition specific barcodes comprise 2 barcodes that are each greater than 2 nucleotides long.

9. The method of claim 8 , wherein all two partition specific barcodes comprise at least two differences in the nucleic acid sequence.

10. The method as in one of claims 1 - 2 wherein the sequence information comprises sequence reads obtained by sequencing.

11. The method as in one of claims 1 - 2 , wherein the sequence information has an average accuracy of greater than 99%.

12. The method as in one of claims 1 - 2 , wherein the sequence information has an average accuracy of greater than 95%.

13. The method as in one of claims 1 - 2 , wherein the sequence information has an average accuracy of greater than 90%.

14. The method as in one of claims 1 - 2 , wherein the sequence information has an average accuracy of greater than 80%.

15. The method of claim 10 , wherein the sequence reads have an accuracy of greater than 99.98%.

16. The method of claim 10 , wherein the sequence information spans more than 50 bp.

17. The method of claim 10 , wherein the sequence information spans more than 100 bp.

18. The method of claim 10 , wherein the sequence information spans more than 200 bp.

19. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 1 month.

20. The method as in one of claims 1 - 2 wherein all sequence information is obtained in less than 2 weeks.

21. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 1 week.

22. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 1 day.

23. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 3 hours.

24. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 1 hour.

25. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 30 minutes.

26. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 10 minutes.

27. The method as in one of claims 1 - 2 , wherein all sequence information is obtained in less than 5 minutes.

28. The method as in one of claims 1 - 2 , wherein at least one target polynucleotide fragment within the sample is flagged if one or more of its sequence segments are substantially similar to one or more sequence segments of a second target polynucleotide fragment partitioned into the same partition.

29. The method as in one of claims 1 - 2 , further comprising a sequence coverage of greater than 20 fold.

30. The method as in one of claims 1 - 2 , further comprising an average sequence coverage of less than 500 fold.

31. The method of claim 10 , wherein the sequencing comprises sequencing by synthesis.

32. The method of claim 10 , wherein the sequencing comprises ion semiconductor sequencing.

33. The method of claim 10 , wherein the sequencing comprises single molecule real time sequencing.

34. The method of claim 10 , wherein the sequencing comprises nanopore sequencing.

35. The method of claim 10 , wherein the sequencing comprises sequencing by electron microscopy.

36. The method as in one of claims 1 - 2 , wherein the plurality of target polynucleotide fragments originates from genomic DNA.

37. The method of claim 36 , wherein the genomic DNA originates from a polyploid genome.

38. The method as in one of claims 1 - 2 , wherein at least one of the target polynucleotide fragments comprises a portion of a major histocompatibility complex gene.

39. The method as in one of claims 1 - 2 , wherein at least one of the target polynucleotide fragments originates from fetal DNA.

40. The method of claim 10 , wherein the sequencing

Assignees

• Pushkarev Dmitry
• Quake Stephen R
• Voskoboynik Ayelet
• Kertesz Michael
• Univ Leland Stanford Junior

Inventors

• Pushkarev Dmitry
• Quake Stephen R
• Voskoboynik Ayelet
• Kertesz Michael

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2535/122

  Massive parallel sequencing · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2527/146

  Concentration of target or template · CPC title