Using non-overlapping fragments for nucleic acid sequencing

• US 8,673,562 B2
• Filed: 07/30/2013
• Issued: 03/18/2014
• 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:
  US13/954,778
• Publication Number:
  US 20130310264A1
• Time in Patent Office: 231 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 determining nucleotide sequences of one or more target polynucleotides of an organism without cell-based cloning, the method comprising the steps of:

providing a plurality of aliquots, most of which contain a mixture of fragments of the target polynucleotide(s), wherein any given segment of the target polynucleotide(s) that is 100 to 300 nucleotides in length is represented by a single non-overlapping fragment in 60% or more of the aliquots containing such segment;

obtaining sequence reads for fragments from the plurality of aliquots;

determining the aliquot of origin of a plurality of fragments from which sequence reads have been obtained; and

assembling the sequence reads to produce an assembled sequence.

2. The method of claim 1, wherein said fragments comprise tags that identify their respective aliquot of origin.

3. The method of claim 2, comprising combining the aliquots to form one or more combined mixtures, and obtaining sequence reads from tagged fragments of the target polynucleotide(s) in the combined mixtures.

4. The method of claim 1, wherein obtaining sequence reads for fragments from the aliquots comprises further fragmenting the fragments in the aliquots to produce subfragments of the target polynucleotide(s), and obtaining sequence reads for the subfragments.

5. The method of claim 4, comprising tagging at least some of the subfragments with oligonucleotide tags that identify the aliquot of origin of the fragment from which each of the subfragments was obtained prior to obtaining the sequence reads.

6. The method of claim 4, comprising tagging the subfragments in the aliquots by way of tagged PCR amplification.

7. The method of claim 1, wherein obtaining sequence reads for fragments from the plurality of aliquots comprises amplifying the fragments in the aliquots to produce amplicons, and obtaining sequence reads for the amplicons.

8. The method of claim 7, comprising tagging the amplicons, whereby the tag identifies the aliquot of origin of the fragment from which an amplicon was amplified.

9. The method of claim 1, wherein obtaining sequence reads for fragments from the plurality of aliquots comprises amplifying the fragments in the aliquots to produce amplicons, fragmenting the amplicons to produce amplicon fragments, and obtaining sequence reads for the amplicon fragments.

10. The method of claim 1, comprising ligating an oligonucleotide tag to at least some of the fragment(s) after the aliquoting such that the tag identifies the respective aliquot of origin.

11. The method of claim 1, wherein the target polynucleotide(s) comprise the genome of an organism.

12. The method of claim 11, wherein the organism is human.

13. The method of claim 1, comprising determining a haplotype of the organism.

14. The method of claim 1, wherein each aliquot comprises less than one genome equivalent of the organism.

15. The method of claim 1, wherein on average, each aliquot comprises between 2/1536 and 50/96 of a coverage amount.

16. The method of claim 1, comprising isolating the target polynucleotide(s) from cells.

17. The method of claim 16, comprising isolating the target polynucleotide(s) from 10 to 30 cells.

18. The method of claim 1, wherein the plurality of aliquots comprises an entire preparation of DNA isolated from a cell population.

19. The method of claim 1, wherein at least sixty percent of the aliquots contain only non-overlapping fragments.

20. The method of claim 1, wherein at least ninety percent of the aliquots contain only non-overlapping fragments.