Genome sequence analysis using tagged amplicons

• US 8,765,382 B2
• Filed: 08/23/2013
• Issued: 07/01/2014
• 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:
  US13/975,223
• Publication Number:
  US 20130345070A1
• Time in Patent Office: 312 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 characterizing the genome 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 contain a mixture of fragments of the genome;

amplifying the fragments in at least some of the aliquots to produce amplicons such that each amplicon comprises a tag sequence whereby two amplicons with the same tag sequence are identified as having the same aliquot of origin;

obtaining nucleic acid sequence reads for the amplicons;

determining amplicons that have the same tag sequence; and

assembling the sequence reads to produce an assembled sequence of the genome.

2. The method of claim 1, comprising further fragmenting the fragments after the aliquoting to produce subfragments.

3. The method of claim 1, comprising amplifying the fragments or subfragments thereof in the aliquots by tagged polymerase chain reaction (PCR).

4. The method of claim 1, comprising ligating an oligonucleotide tag to at least some of the fragments after the aliquoting such that the tag identifies the respective aliquot of origin.

5. The method of claim 1, comprising mixing together tagged amplicons from a plurality of the aliquots before obtaining the sequence reads.

6. The method of claim 1, 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.

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

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

9. The method of claim 1, wherein the step of obtaining nucleic acid sequence reads comprises:

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

obtaining sequence reads from amplicons on the array.

10. The method of claim 9, wherein the random array of the amplicons is fixed to the surface in a pattern.

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

12. The method of claim 1, wherein said fragments of the genome are generated by a process that comprises sonication, treatment with DNase I, or tagged PCR amplification of genomic DNA of the organism.

13. A method of characterizing the genome of an organism without cell-based cloning, the method comprising the steps of:

providing a plurality of aliquots of a sample, each aliquot containing a heterogeneous mixture of polynucleotides comprising different sequences of said genome;

amplifying the polynucleotides in at least some of the aliquots to produce amplicons such that each amplicon comprises a tag sequence whereby two amplicons with the same tag sequence are identified as having the same aliquot of origin;

obtaining nucleic acid sequence reads for the amplicons;

determining amplicons that have the same tag sequence; and

assembling the sequence reads to produce an assembled genomic sequence.

14. The method of claim 13, comprising mixing together tagged amplicons from a plurality of the aliquots before obtaining the sequence reads.