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

Patent metadata

Field	Value
Publication number	US-10227585-B2
Application number	US-201615148928-A
Country	US
Kind code	B2
Filing date	May 6, 2016
Priority date	Sep 12, 2008
Publication date	Mar 12, 2019
Grant date	Mar 12, 2019

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.

First claim

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for preparing a DNA sequencing library, comprising:
(a) circularizing a target fragment library with a plurality of adaptor molecules to produce a population of circularized double-stranded DNA molecules, wherein the adaptor molecules comprise a first defined sequence, a sequence tag, and a second defined sequence such that at least one circularized DNA molecule comprises a sequence tag and a member of the target fragment library;
(b) amplifying the population of circularized double-stranded DNA molecules to produce one or more copies of the circularized DNA molecules, wherein the copies comprise the sequence tag of the parent circularized double-stranded DNA molecule;
(c) fragmenting the plurality of copies of circularized double-stranded DNA molecules to produce a plurality of linear double-stranded DNA molecules; and
(d) adding a third sequence to at least one of a first end and a second end of at least one of the plurality of linear double-stranded DNA molecules from step (c); and
(e) amplifying a region of at least one of the plurality of linear double-stranded DNA molecules using oligonucleotide primers that bind to a portion of the third sequence paired with oligonucleotide primers that bind to a portion of the first defined sequence and/or oligonucleotide primers that bind to a portion of the second defined sequence to produce a plurality of amplicons, wherein at least one amplicon comprises the sequence tag and sequence complementary to a portion of a single member of the target fragment library.

2. The method of claim 1 , wherein the plurality of amplicons comprising the sequence tag further comprise at least one amplicon comprising sequence complementary to a portion of one or both of the first defined sequence and the second defined sequence.

3. The method of claim 1 , wherein the plurality of amplicons comprising the sequence tag further comprise at least one amplicon comprising sequence from at least one of the linear double-stranded DNA molecules, wherein the sequence from at least one of the linear double-stranded DNA molecules is located proximal to the first defined sequence.

4. The method of claim 1 , wherein the plurality of amplicons comprising the sequence tag further comprise at least one amplicon comprising sequence from at least one of the linear double-stranded DNA molecules, wherein the sequence is located proximal to the second defined sequence.

5. The method of claim 1 , wherein said amplifying in step (b) comprises isothermal rolling circle amplification.

6. The method of claim 1 , wherein said amplifying in step (b) comprises multiple displacement amplification.

7. The method of claim 1 , wherein the first and second defined sequence are the same.

8. The method of claim 1 , wherein the first and second defined sequence are different.

9. The method of claim 1 , wherein the adaptor molecules further comprise restriction enzyme sites.

10. The method of claim 1 , wherein the plurality of linear double-strand DNA molecules may be the same or different.

11. The method of claim 1 , wherein the sequence tag is degenerate and each circularized double-stranded DNA molecule and each amplicon derived from that circularized double-stranded DNA molecule contains the same sequence tag and a member of the target fragment library.

12. The method of claim 11 , further comprising sequencing the plurality of amplicons to produce a plurality of associated sequences.

13. The method of claim 12 , further comprising assembling the plurality of associated sequences that have the same sequence tag into longer reads of contiguous sequences derived from the same target fragment.

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

Assignees

• Univ Washington

Inventors

• Shendure Jay
• Hiatt Joseph
• Patwardhan Rupali
• Turner Emily

Classifications

• C12N15/66

  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 title

• C40B40/06

  Libraries containing nucleotides or polynucleotides, or derivatives thereof · CPC title

• C12Q1/6874

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

• C40B50/06

  Biochemical methods, e.g. using enzymes or whole viable microorganisms · CPC title

• C12N15/1093

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title