Error detection in sequence tag directed subassemblies of short sequencing reads

• US 10,577,601 B2
• Filed: 05/12/2017
• Issued: 03/03/2020
• Est. Priority Date: 09/12/2008
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Shendure, Jay, Hiatt, Joseph, Patwardhan, Rupali, Turner, Emily
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/594,476
• Publication Number:
  US 20170247687A1
• Time in Patent Office: 1,026 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The invention provides methods for preparing DNA sequencing libraries by assembling short read sequencing data into longer contiguous sequences for genome assembly, full length cDNA sequencing, metagenomics, and the analysis of repetitive sequences of assembled genomes.

Claims

1. A method for detecting an error occurring in the preparation and/or sequencing of a DNA sequencing library, the method comprising:

(a) incorporating at least one first nucleic acid adaptor molecule into at least one member of a target library comprising a plurality of nucleic acid molecules, wherein the first adaptor molecule comprises a first tag sequence;

(b) amplifying the plurality of nucleic acid molecules to produce an input library comprising a first plurality of amplified DNA molecules, wherein the amplified DNA molecules comprise a sequence identical to or complementary to the first tag sequence and a sequence identical to or complementary to at least a portion of the at least one member of the target library;

(c) sequencing at least a portion of the plurality of amplified DNA molecules to produce a plurality of sequencing reads corresponding to the at least one member of the target library and comprising a sequence identical to or complementary to the first tag sequence;

(d) grouping the plurality of sequencing reads that correspond to the same at least one member of the target library based solely on the commonality of having the first tag sequence or a complement thereof to produce a plurality of grouped sequencing reads; and

(e) detecting whether an error exists at a nucleotide position, wherein an error exists when a variation of nucleotide identity exists among the plurality of grouped sequencing reads at a position corresponding to a nucleotide in the at least one member of the target library.

2. The method of claim 1, wherein the method further comprises determining the correct identity of a nucleotide at the position where the variation of nucleotide identity is detected, wherein the correct identity is determined based on a consensus of individual base calls in the plurality of grouped sequencing reads.

3. The method of claim 2, wherein the consensus of individual base calls is the most common base call at the nucleotide position in the plurality of grouped sequencing reads.

4. The method of claim 1, wherein the method further comprises eliminating from further analysis the identity of the nucleotide at the position in a sequencing read where an error is detected.

5. The method of claim 1, wherein the method further comprises eliminating from further analysis a sequencing read determined to comprise a sequencing error.

6. The method of claim 5, wherein the sequencing read is determined to comprise a sequencing error when it comprises a nucleotide base call that differs from the consensus nucleotide base call provided by the plurality of grouped sequencing reads.

7. The method of claim 1, wherein the first tag sequence comprises a unique nucleotide sequence that distinguishes the at least one member of the target library from other members of the target library.

8. The method of claim 1, further comprising fragmenting at least a portion of the first plurality of amplified DNA molecules in the input library from step (b) to produce a plurality of linear DNA fragments having a first end and a second end.

9. The method of claim 8, further comprising attaching at least one second nucleic acid adaptor molecule to one or both ends of at least one of the plurality of linear DNA fragments, wherein the second adaptor molecule comprises a defined sequence.

10. The method of claim 9, further comprising amplifying the plurality of linear DNA fragments to produce a second plurality of amplified DNA molecules, wherein at least one of the second plurality of amplified DNA molecules comprises a sequence identical to or complementary to the first tag sequence, a sequence identical to or complementary to at least a portion of the second adaptor molecule, and a sequence identical to or complementary to at least a portion of a member of the target library.

11. The method of claim 10, wherein at least a portion of the second plurality of amplified DNA molecules is sequenced in step (c) of the method to produce a plurality of associated sequence reads for each sequenced DNA molecule corresponding to the at least one member of the target library.

12. The method of claim 11, wherein the associated sequence reads comprise a first sequence read and a second sequence read, wherein the first sequence read comprises the first tag sequence of the first adaptor that uniquely identifies a single nucleic acid member of the target library, and wherein the second sequence read comprises a sequence adjacent to the defined sequence of the second adaptor and represents the sequence adjacent to a fragment breakpoint from the fragmented input library.

13. The method of claim 12, wherein a plurality of second sequence reads that are each associated with a first sequence read are grouped in step (d) of the method, wherein the first sequence read contains the first tag sequence identifying a single nucleic acid member of the target library.

14. The method of claim 1, wherein the grouping step (d) comprises generating an alignment of the plurality of sequencing reads.

15. The method of claim 1, wherein the incorporating at least one first nucleic acid adaptor molecule into at least one member of a target library in step (a) results in at least one circular nucleic acid molecule comprising the first nucleic acid adaptor and the member of a target library.

16. A method for correcting an error occurring in the preparation and/or sequencing of a DNA sequencing library, the method comprising:

(a) incorporating at least one first nucleic acid adaptor molecule into at least one member of a target library comprising a plurality of nucleic acid molecules, wherein the first adaptor molecule comprises a first tag sequence;

(b) amplifying the plurality of nucleic acid molecules to produce an input library comprising a first plurality of amplified DNA molecules, wherein the amplified DNA molecules comprise a sequence identical to or complementary to the first tag sequence and a sequence identical to or complementary to at least a portion of the at least one member of the target library;

(c) sequencing at least a portion of the plurality of amplified DNA molecules to produce a plurality of sequencing reads corresponding to the at least one member of the target library and comprising a sequence identical to or complementary to the first tag sequence;

(d) grouping the plurality of sequencing reads that correspond to the at least one member of the target library based solely on the commonality of having the first tag sequence or a complement thereof to produce a plurality of grouped sequencing reads;

(e) detecting whether an error exists at a nucleotide position, wherein an error exists when a variation of nucleotide identity exists among the plurality of grouped sequencing reads at a position corresponding to a nucleotide in the at least one member of the target library; and

(f) determining a correct identity of the nucleotide at the position where the variation of nucleotide identity is detected, wherein the correct identity is determined based on a consensus of individual base calls in the plurality of grouped sequencing reads.

17. The method of claim 16, wherein the consensus of individual base calls is the most common base call at the nucleotide position in the plurality of grouped sequencing reads.

18. A method of detecting an error occurring in the preparation and/or sequencing of a DNA sequencing library, the method comprising:

(a) grouping a plurality of nucleic acid sequencing reads based solely on the commonality of having a first non-degenerate sequence tag to produce a plurality of grouped sequencing reads, wherein the nucleic acid sequencing reads are produced by;

(i) incorporating at least one first nucleic acid adaptor molecule into at least one member of a target library comprising a plurality of nucleic acid molecules, wherein the first adaptor molecule comprises the first tag sequence;

(ii) amplifying the plurality of nucleic acid molecules to produce an input library comprising a first plurality of amplified DNA molecules, wherein the amplified DNA molecules comprise a sequence identical to or complementary to the first tag sequence and a sequence identical to or complementary to at least a portion of the at least one member of the target library; and

(iii) sequencing at least a portion of the plurality of amplified DNA molecules to produce the plurality of sequencing reads that each comprise a sequence identical to or complementary to at least a portion of the at least one member of the target library and a sequence identical to or complementary to the first tag sequence; and

(b) detecting whether an error exists at a nucleotide position, wherein an error exists when a variation of nucleotide identity exists among the plurality of grouped sequencing reads at a position corresponding to a nucleotide in the at least one member of the target library.

19. The method of claim 18, wherein the method further comprises determining a correct identity of the nucleotide at the position where the variation of nucleotide identity is detected, wherein the correct identity is determined based on a consensus of individual base calls in the plurality of grouped sequencing reads.

20. The method of claim 19, wherein the consensus of individual base calls is the most common base call at the nucleotide position in the plurality of grouped sequencing reads.

21. The method of claim 18, wherein the method further comprises eliminating from further analysis the identity of the nucleotide at the position in a sequencing read where an error is detected.

22. The method of claim 18, wherein the method further comprises eliminating from further analysis a sequencing read determined to comprise a sequencing error.

23. The method of claim 18, wherein the grouping step (a) comprises generating an alignment of the plurality of sequencing reads.