Sequencing using a predetermined coverage amount of polynucleotide fragments

• US 8,771,957 B2
• Filed: 08/23/2013
• Issued: 07/08/2014
• Est. Priority Date: 06/15/2005
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Callida Genomics, Inc. (SBH Genomics, Inc.)
• Inventors: Drmanac, Radoje
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/975,215
• Publication Number:
  US 20130345069A1
• Time in Patent Office: 319 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• 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 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 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 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: Y10S 977/778 within specified host or matrix material, e.g. nanocomposite films
• CPC Class Codes: Y10S 977/789 in array format
• CPC Class Codes: Y10S 977/792 : Nucleic acid array, e.g. human genome array
• CPC Class Codes: Y10S 977/88 : with arrangement, process, or apparatus for testing
• CPC Class Codes: Y10S 977/882 : Assembling of separate components, e.g. by attaching

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.

Claims

1. A method of sequencing one or more target polynucleotides, the method comprising the steps of:

providing a predetermined coverage amount of fragments of the one or more target polynucleotides to form a population containing overlapping first-sized fragments of the one or more target polynucleotides;

forming a number of separate mixtures from the population of first-sized fragments;

fragmenting the first-sized fragments in the separate mixtures to form populations of second-sized fragments;

incorporating oligonucleotide tags into the second-sized fragments in the separate mixtures to produce tagged second-sized fragments,

whereby an oligonucleotide tag incorporated into a second-sized fragment in a separate mixture identifies that mixture;

obtaining sequence reads by sequencing at least a portion of a plurality of the tagged second-sized fragments from at least some of the mixtures; and

obtaining complete or partial nucleotide sequences of the one or more target polynucleotides by assembling at least a portion of the sequence reads, wherein the assembling comprises determining that second-sized fragment sequences comprising the same oligonucleotide tag have the same mixture of origin.

2. The method of claim 1, wherein the step of forming mixtures includes replicating the first-sized fragments in the separate mixtures.

3. The method of claim 1, wherein the step of obtaining sequence reads comprises:

generating concatemers that comprise multiple copies of tagged second-sized fragments that substantially cover the first-sized fragments in at least some of the separate mixtures;

forming a random array of the concatemers fixed to a surface at a density such that at least a majority of the concatemers are optically resolvable; and

generating a number of sequence reads from the concatemers,

wherein the number of sequence reads is selected such that the reads substantially cover the first-sized fragments.

4. The method of claim 3, wherein the step of generating the number of sequence reads comprises:

(a) contacting the concatemers with a first set of probes under conditions that permit the formation of perfectly matched duplexes between probes of the first set of probes and complementary sequences on the concatemers;

(b) contacting the concatemers with a second set of probes under conditions that permit the formation of perfectly matched duplexes between probes of the second set of probes and complementary sequences on the concatemers;

(c) producing ligated probes by ligating together pairs of probes hybridized to a concatemer at contiguous sites, wherein the pairs comprise a first probe from the first set of probes and a second probe from the second set of probes;

(d) identifying the sequences of the ligated probes; and

(e) repeating steps (a) through (d) until the sequences of the first-sized fragments is substantially determined from the identities of the sequences of the ligated probes.

5. The method of claim 1, comprising combining the separate mixtures to form one or more combined mixtures, and obtaining sequence reads from tagged second-sized fragments or from subfragments of the tagged second-sized fragments of the target polynucleotide(s) in the combined mixtures.

6. The method of claim 1, comprising determining a haplotype of an organism.

7. The method of claim 1, comprising determining a genetic variation associated with a disease.

8. The method of claim 1, wherein the target polynucleotide is genomic DNA and the first-sized fragments are generated by sonication, treatment with DNase I, or tagged PCR amplification of said genomic DNA.

9. The method of claim 1, wherein the number of separate mixtures is selected so that the probability of overlapping first-sized fragments in any one of the mixtures is less than 10%.

10. The method of claim 1, wherein the number of separate mixtures is selected so that the probability of overlapping first-sized fragments in any one of the mixtures is less than 1%.

11. The method of claim 1, wherein the number of separate mixtures is selected so that the probability of overlapping first-sized fragments in any one of the mixtures is less than 0.01%.

12. The method of claim 1, wherein the steps of fragmenting the first-sized fragments and incorporating the oligonucleotide tags are accomplished by amplifying the first-sized fragments or subfragments thereof by tagged PCR amplification so as to produce the tagged second-sized fragments.

13. A method of determining a nucleotide sequence of one or more target polynucleotides of an organism without cell-based cloning, the method comprising the steps of:

providing a plurality of aliquots of a sample, at least some of which comprise a plurality of first-sized fragments of the target polynucleotide(s);

producing tagged second-sized fragments from the first-sized fragments;

mixing together fragments from a plurality of the aliquots to form a mixture;

obtaining sequence reads from second-sized fragments of the mixture; and

assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s) by a process that comprises grouping sequence reads from fragments that have the same tag.

14. The method of claim 13, wherein least ninety percent of the aliquots containing a plurality of first-sized fragments contain only non-overlapping fragments.

15. The method of claim 13, comprising determining a haplotype of an organism.

16. The method of claim 13, wherein the step of providing a plurality of aliquots of a sample comprises obtaining a nucleic acid-containing biological sample from an animal and dividing the sample into a plurality of aliquots.

17. The method of claim 16, wherein the nucleic acid is genomic DNA of the animal.

18. The method of claim 17, wherein the animal is a human.

19. A method of determining a nucleotide sequence of one or more target polynucleotides of an organism without cell-based cloning, the method comprising the steps of:

providing a plurality of aliquots, at least some of which comprise a plurality of first-sized fragments of the target polynucleotide(s) wherein any given portion of the target polynucleotide(s) is represented by a single non-overlapping fragment in sixty percent or more of the mixtures containing such portion;

producing tagged second-sized fragments from the first-sized fragments;

mixing together second-sized fragments from a plurality of the aliquots to form a mixture;

obtaining sequence reads from second-sized fragments of the mixture; and

assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s) by a process that comprises grouping sequence reads from fragments that have the same tag.