Nucleic acid analysis by random mixtures of non-overlapping fragments

Patent metadata

Field	Value
Publication number	US-11414702-B2
Application number	US-201916730829-A
Country	US
Kind code	B2
Filing date	Dec 30, 2019
Priority date	Jun 15, 2005
Publication date	Aug 16, 2022
Grant date	Aug 16, 2022

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 sequencing a target polynucleotide, the method comprising the steps of:
obtaining a population of nucleic acids containing overlapping first-sized fragments of the target polynucleotide;
forming a mixture containing labeled second-sized fragments from the first-sized fragments, wherein:
(1) each second-sized fragment contains a shorter portion of the target polynucleotide than the first-sized fragment from which it was obtained, labeled with an oligonucleotide tag,
(2) at least 96 of the second-sized fragments in the mixture contain different portions of the target polynucleotide labeled with different oligonucleotide tags, and
(3) some of the second-sized fragments contain different target polynucleotide fragments labeled with the same oligonucleotide tag, whereby second-sized fragments labeled with the same oligonucleotide tag more often contain fragment sequences that occur within 100 kb of each other in the target polynucleotide, compared with second-sized fragments labeled with different oligonucleotide tags;
obtaining sequence reads from at least some of the tagged second-sized fragments in the mixture; and
obtaining complete or partial nucleotide sequences of the target polynucleotide by a process that comprises ordering and assembling sequence reads whereby reads that contain the same oligonucleotide tag sequences are grouped together.

2. The method of claim 1 , wherein the sequence reads that are obtained for the second-sized fragments are 100 to 1,000 nucleotides in length, each containing a part of the sequence of the target polynucleotide and the sequence of the respective oligonucleotide tag.

3. The method of claim 1 , wherein the median length of the second-sized fragments is 30 to 3,000 bases.

4. The method of claim 1 , wherein the probability of second-sized fragments in the mixture containing fragments of the target polynucleotide having overlapping sequences labeled with the same oligonucleotide tag is less than 1%.

5. The method of claim 1 , wherein the second-sized fragments contain two fragments that have sequences that are nearby but not immediately beside each other in the target polynucleotide separated by DNA that contains a common adaptor sequence.

6. The method of claim 1 , wherein the target polynucleotide is genomic DNA.

7. The method of claim 1 , wherein the mixture comprises 10-30 equivalents of the target polynucleotide.

8. The method of claim 1 , wherein the target polynucleotide comprises genomic DNA from multiple organisms.

9. The method of claim 1 , wherein the target polynucleotide is a cDNA expression library obtained by reverse-transcribing mRNA from a biological sample.

10. The method of claim 1 , wherein the sequence reads are obtained by a process that comprises sequencing by synthesis.

11. The method of claim 1 , wherein different oligonucleotide tags are used to label second-sized fragments in each of a plurality of aliquots, which are then pooled to form said mixture.

12. The method of claim 1 , wherein the second-sized fragments labeled with an oligonucleotide tag are obtained from the first-sized fragments by a process that comprises replicating the first-sized fragments with tagged primers.

13. A method of sequencing a genome, comprising the steps of:
obtaining a population of nucleic acids containing overlapping first-sized fragments of the genome;
forming a mixture containing labeled second-sized fragments from the first-sized fragments, wherein:
(1) each second-sized fragment contains a shorter portion of the genome than the first-sized fragment from which it was obtained, labeled with an oligonucleotide tag,
(2) at least 96 of the second-sized fragments in the mixture contain different portions of the genome labeled with different oligonucleotide tags, and
(3) some of the second-sized fragments contain different genome fragments labeled with the same oligonucleotide tag, whereby second-sized fragments labeled with the same oligonucleotide tag more often contain fragment sequences that occur within 100 kb of each other in the genome, compared with second-sized fragments labeled with different oligonucleotide tags;
obtaining sequence reads from at least some of the tagged second-sized fragments in the mixture; and
obtaining complete or partial nucleotide sequences of the genome by a process that comprises ordering and assembling sequence reads whereby reads that contain the same oligonucleotide tag sequences are grouped together.

14. The method of claim 13 , wherein the genome is a mammalian genome.

15. The method of claim 14 , wherein the sequence reads that are obtained for the second-sized fragments are 100 to 1,000 nucleotides in length, each containing a part of the sequence of the mammalian genome and the sequence of the respective oligonucleotide tag.

16. The method of claim 14 , wherein the median length of the second-sized fragments is 30 to 3,000 bases.

17. The method of claim 14 , wherein the probability of second-sized fragments in the mixture containing fragments of the genome having overlapping sequences labeled with the same oligonucleotide tag is less than 1%.

18. The method of claim 14 , wherein the sequence reads are obtained by a process that comprises sequencing by synthesis.

19. The method of claim 14 , wherein different oligonucleotide tags are used to label second-sized fragments in each of a plurality of aliquots, which are then pooled to form said mixture.

20. The method of claim 14 , wherein the second-sized fragments labeled with an oligonucleotide tag are obtained from the first-sized fragments by a process that comprises replicating the first-sized fragments with tagged primers.

Assignees

• Complete Genomics Inc

Inventors

• Drmanac Radoje

Classifications

• Y10S977/88

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

• Y10S977/778

  within specified host or matrix material, e.g. nanocomposite films · CPC title

• C12Q2531/125

  Rolling circle · CPC title

• Y10S977/789

  in array format · CPC title

• G01N15/1404

  Handling flow, e.g. hydrodynamic focusing · CPC title