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

• US 8,383,345 B2
• Filed: 09/14/2009
• Issued: 02/26/2013
• 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
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US12/559,124
• Publication Number:
  US 20100069263A1
• Time in Patent Office: 1,261 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• 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 for assembling short read sequencing data into longer contiguous sequences and providing for genome assembly, full length cDNA sequencing, metagenomics, and the analysis of repetitive sequences of assembled genomes. The methods generally comprise incorporating adaptor or vector sequences into at least one member of a target fragment library, amplifying the population of target fragment library members to produce a plurality of copies of amplified DNA molecules, fragmenting the plurality of amplified DNA molecules, incorporating additional adaptor sequences, and amplifying a region of at least one of the plurality of amplified DNA molecules. At least one resulting amplicon comprises a sequence identical to or complementary to at least a portion of a sequence tag and a sequence identical to or complementary to a portion of a target fragment library member.

Claims

1. A method for preparing a DNA sequencing library, 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 at least a portion of 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 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 at least one member of the target library;

(c) fragmenting the input library to produce a plurality of linear DNA fragments having a first end and a second end;

(d) 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 at least a portion of the second adaptor molecule comprises a second defined sequence; and

(e) amplifying the plurality of linear DNA fragments to produce a sequencing library comprising a second plurality of amplified DNA molecules, wherein at least one of the plurality of amplified DNA molecules comprises sequence identical to or complementary to at least a portion of the first adaptor molecule, sequence identical to or complementary to at least a portion of the second adaptor molecule, and sequence identical to or complementary to at least a portion of a member of the target library.

2. The method of claim 1, wherein the at least one first nucleic acid adaptor is attached to or incorporated at one or both ends of the at least one the target library molecule.

3. The method of claim 1, wherein step (a) comprises circularizing a plurality of the target library nucleic acid molecules with a plurality of first adaptor molecules, wherein the first adaptor molecules comprise a first defined sequence and a degenerate sequence tag.

4. The method of claim 3, wherein the first defined sequence comprises a first restriction enzyme recognition site.

5. The method of claim 3, wherein the first adaptor molecules further comprise a second defined sequence.

6. The method of claim 5, wherein the second defined sequence comprises a second restriction enzyme recognition site.

7. The method of claim 1, wherein step (a) further comprises 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 the first 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.

8. The method of claim 1, wherein the amplification in step (b) utilizes at least one oligonucleotide primer comprising a sequence complementary to at least a portion of the first adaptor sequence.

9. The method of claim 1, wherein the amplification in step (e) utilizes at least one oligonucleotide primer comprising a sequence complementary to at least a portion of the second adaptor sequence.

10. The method of claim 1, wherein the second plurality of amplified DNA molecules are size selected in a range of about 300 to about 600 base pairs.

11. The method of claim 1, further comprising randomly ligating together members of the first plurality of amplified DNA molecules to produce the input library comprising concatemer molecules, wherein the concatamers comprise a plurality of target library molecules having adaptor molecules attached thereto or incorporated therein.

12. The method of claim 1, further comprising sequencing at least a portion of the second plurality of amplified DNA molecules to produce a plurality of associated sequences for each sequenced amplified DNA molecule.

13. The method of claim 12, wherein the second plurality of amplified DNA molecules are sequenced using a first oligonucleotide primer that is complementary to the sense or antisense strand of the first defined sequence of the first adaptor and a second oligonucleotide primer that is complementary to the sense or antisense strand of the second defined sequence of the second adaptor to produce the plurality of associated sequences.

14. The method of claim 12, wherein the associated sequences comprise a first sequence and a second sequence, wherein the first sequence comprises a sequence adjacent to the first defined sequence of the first adaptor and 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 12, wherein the method further comprises assembling the plurality of associated sequences that are associated with the same nucleic acid member of the target library to generate one or more longer sequences of the nucleic acid member.

16. The method of claim 14, wherein the method further comprises assembling the plurality of second sequences that are associated with the same uniquely identifying sequence in the plurality of first sequences, thereby generating one or more longer sequences of the nucleic acid member of the target library comprising the associated fragmentation breakpoint sequences from the plurality associated second sequences.

17. The method of claim 1, wherein each member of the target library is about 300 base pairs to about 10 kilobase pairs in length.

18. A method for preparing a DNA sequencing library, comprising:

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

(b) incorporating second nucleic acid tag adaptor molecule into the second end of the at least one linear member of the target library, wherein at least a portion of the second tag adaptor molecule comprises a second defined tag sequence;

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

(d) fragmenting the input library to produce a plurality of linear DNA fragments having a first end and a second end;

(e) attaching at least one nucleic acid shotgun adaptor molecule to one or both ends of at least one of the plurality of linear DNA fragments, wherein at least a portion of the shotgun adaptor molecule comprises a defined shotgun sequence; and

(f) amplifying the plurality of linear DNA fragments to produce a sequencing library comprising a second plurality of amplified DNA molecules, wherein at least one of the plurality of amplified DNA molecules comprises sequence identical to or complementary to at least a portion of the first to adaptor molecule, sequence identical to or complementary to at least a portion of the shotgun adaptor molecule, and sequence identical to or complementary to at least a portion of a member of the target library, and wherein at least one of the plurality of amplified DNA molecules comprises sequence identical to or complementary to at least a portion of the second tag adaptor molecule, sequence identical to or complementary to at least a portion of the shotgun adaptor molecule, and sequence identical to or complementary to at least a portion of a member of the target library.

19. The method of claim 18, wherein the first defined tag sequence is different from the second defined tag sequence.

20. The method of claim 18, wherein the amplification in step (c) is performed using the polymerase chain reaction (PCR), wherein a first amplification primer comprises at least a portion of the first tag adaptor sequence or its complement, and wherein a second amplification primer comprises at least a portion of the second tag adaptor sequence or its complement.

21. The method of claim 18, wherein the first plurality of amplified DNA molecules are randomly ligated together to produce concatemer molecules.

22. The method of claim 18, wherein the amplification in step (f) is performed by PCR using two sets of primer pairs in separate reactions.

23. The method of claim 22, wherein the primer pair of the first PCR includes a first amplification primer comprising at least a portion of the first tag adaptor sequence or its complement and a second amplification primer comprising at least a portion of the shotgun adaptor sequence or its complement.

24. The method of claim 22, wherein the primer pair of the second PCR includes a first amplification primer comprising at least a portion of the second tag adaptor sequence or its complement and a second amplification primer comprising at least a portion of the shotgun adaptor sequence or its complement.

25. The method of claim 18, further comprising sequencing at least a portion of the second plurality of amplified DNA molecules to produce a plurality of associated sequences for each amplified DNA molecule.

26. The method of claim 25, wherein the second plurality of amplified DNA molecules are sequenced using a first sequencing primer that is complementary to the sense or antisense strand of the first or second defined tag sequence to produce first or second tag reads and a second sequencing primer that is complementary to the sense or antisense strand of the defined shotgun sequence of the shotgun adaptor to produce shotgun reads.

27. The method of claim 18, further comprising assembling the plurality of shotgun sequences that are associated with the same nucleic acid member of the target library as determined by association with a common first or second tag read to generate one or more longer sequences of the nucleic acid member of the target library.

28. The method of claim 27, further comprising assembling multiple longer sequences of the nucleic acid member associated with the first and second tag reads of the same nucleic acid member of the target library.