SEQUENCE TAG DIRECTED SUBASSEMBLY OF SHORT SEQUENCING READS INTO LONG SEQUENCING READS

• US 20130137588A1
• Filed: 02/06/2013
• Published: 05/30/2013
• 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
• Granted Patent: US 8,865,410 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 compositions and methods for preparing DNA sequencing libraries. In particular, the method relates to preparing DNA sequencing libraries from kilobase scale nucleic acids. The invention also provides methods for assembling short read sequencing data into longer contiguous sequences. The method is useful for various applications in genomics, including genome assembly, full length cDNA sequencing, metagenomics, and the analysis of repetitive sequences of assembled genomes.

Claims

1. A method for detecting errors 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 defined 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 molecules comprise a sequence identical to or complementary to at least a portion of the first adaptor molecule and 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;

(d) grouping the plurality of sequencing reads that correspond to the at least one member of the target library; and

(e) detecting whether an error exists at a nucleotide position, wherein an error exists when variation of nucleotide identity exists among the 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 the nucleotide at the position where variation of nucleotide identity is detected, wherein the correct identity is determined as the most probable base call based on the consensus of the 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 3, wherein the consensus of individual base calls in the plurality of grouped sequencing reads is determined considering unambiguous base calls in the plurality of grouped sequencing reads at the nucleotide position.

5. 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.

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

7. The method of claim 6, 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 grouped plurality of sequencing reads.

8. The method of claim 1, wherein the plurality of sequencing reads that correspond to the at least one member of the target library comprise a common tag sequence, whereby the plurality of sequencing reads are grouped based on having the common sequence tag.

9. The method of claim 8, wherein the common tag sequence is derived from a source selected from the group consisting of a unique nucleotide sequence in the incorporated first adaptor molecule and an endogenous nucleotide sequence in the at least one member of the target library adjacent to the incorporated first nucleic acid adaptor molecule.

10. 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.

11. The method of claim 10, 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 second defined sequence.

12. The method of claim 11, 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 at least a portion of the first adaptor molecule, 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.

13. The method of claim 12, 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.

14. The method of claim 13, wherein the associated sequences comprise a first sequence read and a second sequence read, wherein the first sequence comprises a tag sequence adjacent to the first defined sequence of the first adaptor that uniquely identifies a single nucleic acid member of the target library, and wherein the second sequence comprises a sequence adjacent to the second defined sequence of the second adaptor and represents the sequence adjacent to a fragment breakpoint from the fragmented input library.

15. The method of claim 13, where in the tag sequence adjacent to the first defined sequence of the first adaptor is derived from a source selected from the group consisting of a unique nucleotide sequence in the first adaptor molecule and an endogenous nucleotide sequence in the at least one member of the target library adjacent to the incorporated first nucleic acid adaptor molecule.

16. The method of claim 14, wherein a plurality of second sequence reads that are each associated with a first sequence read containing a common tag sequence identifying a single nucleic acid member of the target library, whereby the plurality of second sequence reads are grouped in step (d) of the method.

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

18. 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 molecules comprising the first nucleic acid adaptor and the member of a target library.

19. A method for correcting errors 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 defined 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 molecules comprise a sequence identical to or complementary to at least a portion of the first adaptor molecule 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;

(d) grouping the plurality of sequencing reads that correspond to the at least one member of the target library;

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

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

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