Sequence tag directed subassembly of short sequencing reads into long sequencing reads

• US 8,846,347 B2
• Filed: 02/05/2013
• Issued: 09/30/2014
• 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
• Application Number:
  US13/759,292
• Publication Number:
  US 20130137605A1
• Time in Patent Office: 602 Days
• Field of Search: 435/91.1, 435/91.2
• US Class Current: 435/91.1
• 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 generating sequence assemblies from short sequencing reads, comprising:

a) introducing a fragmentation breakpoint in at least one member of an input library to produce a plurality of linear DNA fragments, wherein a first fragment end and a second fragment end are proximal to the fragmentation breakpoint,

b) attaching common nucleic acid adaptors to the first fragment end and the second fragment end proximal to the fragmentation breakpoint, wherein the common adaptors attached to the first fragment end and the second fragment end comprise the same unique sequence tag,

c) optionally amplifying the plurality of linear DNA fragments to produce a sequencing library comprising a plurality of amplified DNA fragments, wherein at least one of the plurality of amplified DNA fragments comprises;

i) sequence complementary to at least the unique sequence tag of an adaptor, and

ii) sequence complementary to at least a portion of a member of the input library,

d) sequencing at least a portion of the DNA fragments, wherein the presence of a unique adaptor sequence tag in a plurality of fragment sequences thereby associates the fragment sequences having ends that were proximal to the same fragmentation breakpoint, and

e) assembling the plurality of breakpoint-associated fragment sequences to generate longer subassembly sequences of the input library.

2. The method of claim 1, wherein prior to step a) at least one of a plurality of nucleic acid end adaptors is attached to one or both ends of at least one member of a target library comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid end adaptors comprise a first defined sequence.

3. The method of claim 2, wherein the plurality of nucleic acid end adaptors comprise a degenerate sequence tag.

4. The method of claim 2, wherein a plurality of the target library nucleic acid molecules is circularized with the plurality of nucleic acid end adaptors.

5. The method of claim 4, wherein the nucleic acid end adaptors comprise a degenerate sequence tag.

6. The method of claim 4, wherein the first defined sequence comprises a first restriction enzyme recognition site.

7. The method of claim 4, wherein the nucleic acid end adaptors further comprise a second defined sequence.

8. The method of claim 7, wherein the second defined sequence comprises a second restriction enzyme recognition site.

9. The method of claim 2, further comprising incorporating the at least one nucleic acid member of the target library into a vector to produce at least one circular double stranded DNA molecule, wherein the vector comprises a first end with first nucleic acid end adaptor sequence, and wherein said first end is joined to a first end of the at least one nucleic acid member of the target library.

10. The method of claim 2, further comprising amplifying at least one target nucleic acid molecule to produce an input library comprising a plurality of amplified DNA input molecules, wherein the amplified DNA input molecules comprise sequence identical to or complementary to at least a portion of a nucleic acid end adaptor and sequence identical to or complementary to at least a portion of the at least one member of the target library.

11. The method of claim 10, wherein the amplification comprises use of at least one oligonucleotide primer comprising a sequence complementary to at least a portion of the nucleic acid end adaptor sequence.

12. The method of claim 10, further comprising randomly ligating together members of the plurality of amplified DNA input molecules to produce the input library comprising concatemer molecules, wherein the concatamers comprise a plurality of target DNA molecules having nucleic acid end adaptors attached thereto or incorporated therein.

13. The method of claim 1, wherein the unique sequence tag of the common nucleic acid adaptors attached to the DNA fragment ends proximal to one fragmentation breakpoint is different from the unique sequence tag of common nucleic acid adaptors attached to the fragments ends proximal to a different fragmentation breakpoint.

14. The method of claim 1, wherein step c) is not optional.

15. The method of claim 1, wherein the portion of the DNA fragments are sequenced in step d) using a first oligonucleotide primer that anneals to a portion of the common nucleic acid adaptor or its complement and results in sequencing reads comprising sequence corresponding to the unique sequence tag and sequence of the linear DNA fragment proximal to the fragmentation breakpoint.

16. The method of claim 2, wherein the portion of the DNA fragments in step 1(d) are sequenced using a first oligonucleotide primer that anneals to a portion of the common nucleic acid adaptor or its compliment and a second oligonucleotide that anneals to the defined sequence of the nucleic acid adaptor or its complement, to produce a plurality of end tag associated sequences from each sequenced DNA fragment.

17. The method of claim 16, further comprising assembling the plurality of end tag-associated sequences that are associated with the same end tag that identifies a single nucleic acid member of the target library to generate one or more longer subassembly sequences of the target library member.

18. The method of claim 16, wherein the plurality of end tag-associated sequences comprise a first and second sequence, wherein the first sequence comprises:

1) sequence corresponding to a portion of the end adaptor, and/or

     2) sequence of the DNA fragment adjacent to the end adaptor, one of which uniquely identifies a single nucleic acid member of the target library; and

wherein the second sequence comprises

     1) sequence corresponding to the unique sequence tag of a common nucleic acid adaptor, and

     2) sequence of the DNA fragment corresponding to sequence of the fragmented input library member adjacent to the fragmentation breakpoint.

19. The method of claim 18, further comprising assembling the plurality of second sequences that are associated with the same sequence uniquely identifying the single nucleic acid member of the target library, thereby generating one or more longer subassembly sequences of the target library member and comprising breakpoint-associated sequences.

20. The method of claim 2, wherein each molecule of the target library is about 300 base pairs to about 10 kilobase pairs in length.