Method for sequencing polynucleotides by forming separate fragment mixtures

• US 8,765,375 B2
• Filed: 08/08/2013
• 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:
  US13/962,893
• Publication Number:
  US 20130316920A1
• Time in Patent Office: 327 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 sequencing one or more target polynucleotides, the method comprising the steps of:

fragmenting a predetermined coverage amount of the one or more target polynucleotides to form a population containing overlapping fragments;

forming a number of separate mixtures of origin from the population wherein at least sixty percent of the separate mixtures contain only non-overlapping fragments, and optionally subfragmenting at least some of the fragments in said mixtures, thereby forming subfragments,

wherein the respective mixture of origin of the fragments or subfragments is determinable;

then

obtaining nucleic acid sequence reads for at least a portion of a plurality of the fragments or subfragments; and

obtaining complete or partial nucleotide sequences of the one or more target polynucleotides by assembling sequence reads for the fragments or subfragments, wherein the assembling comprises determining the mixtures of origin of the fragments or subfragments from which the sequence reads were obtained.

2. The method of claim 1, wherein said step of forming includes replicating said fragments in said separate mixtures.

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

generating a plurality of concatemers from at least some of said fragments or subfragments, wherein each concatemer comprises multiple copies of the fragment or subfragment, and wherein the plurality of concatemers substantially covers the target polynucleotide(s);

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

generating sequence reads from the concatemers.

4. The method of claim 3, wherein the step of obtaining nucleic acid 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 said 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) to determine sequence information from at least a portion of one or more fragments or subfragments in the mixtures.

5. The method of claim 1, wherein the fragments or subfragments comprise tags that identify their respective mixture of origin.

6. The method of claim 5, comprising combining the plurality of aliquots to form one or more combined mixtures, and obtaining sequence reads from tagged fragments or tagged subfragments of the target polynucleotide(s) in the combined mixture(s).

7. The method of claim 1, comprising tagging the fragments or subfragments, whereby the tags identify the mixture of origin of the respective fragment or subfragment.

8. The method of claim 7, comprising ligating an oligonucleotide tag to at least some of the fragments or subfragments after formation of the separate mixtures such that the tag identifies the respective mixture of origin.

9. The method of claim 1, further comprising amplifying the fragments or subfragments thereof in the separate mixtures.

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

11. The method of claim 1, wherein at least eighty percent of the separate mixtures contain only non-overlapping fragments.

12. The method of claim 1, wherein at least ninety percent of the separate mixtures contain only non-overlapping fragments.

13. The method of claim 1, wherein after fragmenting and before formation of separate mixtures, the fragments are replicated.

14. The method of claim 1 wherein the mixtures are contained in wells of a multi-well plate.

15. The method of claim 1, wherein said fragmenting comprises sonication, treatment with DNase I, and/or tagged PCR amplification of said one or more target polynucleotides.