Preparing a DNA fragment library for sequencing using tagged primers

Patent metadata

Field	Value
Publication number	US-10125392-B2
Application number	US-201313971797-A
Country	US
Kind code	B2
Filing date	Aug 20, 2013
Priority date	Jun 15, 2005
Publication date	Nov 13, 2018
Grant date	Nov 13, 2018

Abstract

The invention provides methods and kits for ordering sequence information derived from one or more target polynucleotides. In one aspect, one or more tiers or levels of fragmentation and aliquoting are generated, after which sequence information is obtained from fragments in a final level or tier. Each fragment in such final tier is from a particular aliquot, which, in turn, is from a particular aliquot of a prior tier, and so on. For every fragment of an aliquot in the final tier, the aliquots from which it was derived at every prior tier is known, or can be discerned. Thus, identical sequences from overlapping fragments from different aliquots can be distinguished and grouped as being derived from the same or different fragments from prior tiers. When the fragments in the final tier are sequenced, overlapping sequence regions of fragments in different aliquots are used to register the fragments so that non-overlapping regions are ordered. In one aspect, this process is carried out in a hierarchical fashion until the one or more target polynucleotides are characterized, e.g. by their nucleic acid sequences, or by an ordering of sequence segments, or by an ordering of single nucleotide polymorphisms (SNPs), or the like.

First claim

The invention claimed is:

1. A method of preparing a tagged fragment library from a genome, comprising:
a) providing a plurality of aliquots, at least some of which contain a plurality of first polynucleotides, wherein each first polynucleotide in an aliquot comprises a nucleotide sequence from a portion of the genome, and wherein, in at least sixty percent of the aliquots that contain first polynucleotides, the first polynucleotides do not comprise overlapping portions of the genome;
b) replicating at least part of the nucleotide sequence of the first polynucleotides in at least some of the aliquots using tagged primers comprising a tag sequence, thereby producing tagged fragments in said aliquots, wherein the average fragment length of the tagged fragments in an aliquot is less than the length of the first polvnucleotides in the aliquot, and wherein the tagged fragments in different aliquots are labeled with different tag sequences; and
c) combining tagged fragments from the aliquots in step (b) together into a combined mixture, thereby producing the tagged fragment library,
wherein the tagged primers in each aliquot comprise (i) a fragment binding portion that hybridizes to the first polynucleotide(s) in the aliquot and (ii) a tagportion that comprises the tag sequence.

2. The method of claim 1 , further comprising obtaining sequence information from each of a plurality of the tagged fragments in the combined mixture,
wherein the sequence information obtained from each tagged fragment includes genomic sequence and a tag sequence.

3. A method of preparing a tagged fragment library from a genome, comprising:
a) providing a plurality of aliquots, at least some of which contain one or more first polynucleotides, wherein each first polynucleotide in an aliquot comprises a nucleotide sequence from a portion of the genome, and wherein, in at least sixty percent of the aliquots that contain first polynucleotides, the first polynucleotides do not comprise overlapping portions of the genome;
b) replicating at least part of the nucleotide sequence of the first polynucleotides in at least some of the aliquots using tagged primers comprising a tag sequence, thereby producing tagged fragments in said aliquots, wherein the average fragment length of the tagged fragments in an aliquot is less than the length of the first polynucleotides in the aliquot, and wherein the tagged fragments in different aliquots are labeled with different tag sequences; and
c) combining tagged fragments from the aliquots in step (h) together into a combined mixture, thereby producing the tagged fragment library,
wherein the tagged primers in each aliquot comprise (i) a fragment binding portion that hybridizes to the genomic nucleotide sequence in the first polynucleotide(s) in the aliquot and (ii) a tag portion that comprises the tag sequence.

4. The method of claim 3 , further comprising obtaining sequence information from each of a plurality of the tagged fragments in the combined mixture,
wherein the sequence information obtained from each tagged fragment includes genomic sequence and a tag sequence.

5. A method of preparing a tagged fragment library from a genome, comprising:
a) providing a plurality of aliquots, at least some of which contain one or more first polynucleotides, wherein each first polynucleotide in an aliquot comprises a nucleotide sequence from a portion of the genome,, and wherein, in at least sixty percent of the aliquots that contain first polynucleotides, the first polynucleotides do not comprise overlapping portions of the genome;
b) replicating at least part of the nucleotide sequence of the first polynucleotides in at least some of the aliquots using tagged primers comprising a tag sequence, thereby producing tagged fragments in said aliquots, wherein the average fragment length of the tagged fragments in an aliquot is less than the length of the first polynucleotides in the aliquot, and wherein the tagged fragments in different aliquots are labeled with different tag sequences; and
c) combining tagged fragments from the aliquots in step (b) together into a combined mixture, thereby producing the tagged fragment library,
wherein the tagged primers in each aliquot comprise (i) a fragment binding portion that hybridizes to the first polynucleotide(s) in the aliquot and (ii) a tag portion that comprises the tag sequence,
wherein the fragment binding portion of each tagged primer comprises a random sequence.

6. The method of claim 5 , further comprising obtaining sequence information from each of a plurality of the tagged fragments in the combined mixture,
wherein the sequence information obtained from each tagged fragment includes genomic sequence and a tag sequence.

7. The method of claim 1 , wherein the fragment binding portion of each tagged primer in each aliquot comprises a random sequence.

8. The method of claim 3 , wherein the fragment binding portion of each tagged primer in each aliquot comprises a random sequence.

9. The method of claim 7 or claim 8 , wherein the random sequence is a sequence of 6 to 18 nucleotides.

10. The method of claim 1 or claim 5 , wherein the fragment binding portions of the tagged primers in each aliquot hybridize to the genomic nucleotide sequence in the first polynucleotide(s) in the aliquot.

11. The method of claim 3 or claim 5 , wherein at least some of the aliquots provided in step (a) contain a plurality of first polynucleotides.

12. The method of claim 1 , claim 3 , or claim 5 , wherein in at least ninety percent of the aliquots the nucleotide sequence from any given portion of the genome is represented in only a single first polynucleotide in the aliquot.

13. The method of claim 1 , claim 3 , or claim 5 , wherein the plurality of aliquots in (a) comprise at least 10 genome equivalents of DNA, and the number of aliquots in (a) is selected so that the probability of overlapping fragments in individual aliquots of the plurality is less than 0.1%.

14. The method of claim 1 , claim 3 , or claim 5 , wherein the number of aliquots is at least 1536.

15. The method of claim 2 , claim 4 , or claim 6 , wherein the step of obtaining the sequence information comprises:
arraying the tagged fragments on a surface such that a majority of the fragments are optically resolvable;
optionally amplifying the tagged fragments before or after the arraying; and
obtaining sequence reads from the tagged fragments on the surface.

16. The method of claim 2 , wherein the step of obtaining the sequence information includes sequencing by probe ligation.

17. The method of claim 2 , claim 4 , or claim 6 , wherein the step of obtaining the sequence information includes sequencing by synthesis.

18. The method of claim 1 , claim 3 , or claim 5 , wherein the genome is a mammalian genome.

Assignees

• Complete Genomics Inc

Inventors

• Drmanac Radoje

Classifications

• Y10S977/789

  in array format · CPC title

• C07K1/047

  Simultaneous synthesis of different peptide species; Peptide libraries · CPC title

• C12Q1/682

  Signal amplification · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• Y10S977/88

  with arrangement, process, or apparatus for testing · CPC title