Nucleic acid sequence analysis from combined mixtures of amplified fragments

• US 8,765,379 B2
• Filed: 01/31/2011
• 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:
  US13/017,244
• Publication Number:
  US 20110319281A1
• Time in Patent Office: 1,247 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 determining a nucleotide sequence of a genome of an organism, the method comprising the steps of:

aliquotinq fragments obtained from the genome to produce aliquots at least some of which comprise a plurality of the fragments;

amplifying the fragments to produce amplicons having a tag that identifies the aliquot of origin of such amplicon;

mixing together amplicons from the plurality of aliquots to produce a mixture containing amplicons;

obtaining sequence reads from amplicons from the mixture;

determining the tags corresponding to the sequence reads; and

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

2. A method of determining a nucleotide sequence of a genome of an organism, 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 genome;

fragmenting the first-sized fragments to produce second-sized fragments;

amplifying the second-sized fragments to produce amplicons, wherein each amplicon has a tag that identifies the aliquot of origin of such amplicon;

mixing together amplicons from the plurality of aliquots to produce a mixture containing amplicons;

obtaining sequence reads from amplicons from the mixture;

determining the respective aliquot of origin corresponding to the sequence reads; and

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

3. A method of determining a nucleotide sequence of one or more target polynucleotides of an organism, the method comprising the steps of:

aliquoting fragments obtained from the polynucleotide(s) to produce aliquots at least some of which comprise a plurality of the fragments each having a tag;

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

obtaining sequence reads from the fragments of the mixture;

determining the tags corresponding to the sequence reads; and

assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s).

4. A method of determining a nucleotide sequence of one or more target polynucleotides of an organism, the method comprising the steps of:

aliquoting a coverage amount of fragments of the target polynucleotide(s) into a plurality of aliquots that contain multiple different fragments, wherein the coverage amount is in the range of 2 to 50, and the number of aliquots is selected to minimize the probability of having overlapping fragments within the same aliquot;

obtaining sequence reads of the fragments from the plurality of aliquots;

determining the respective aliquot of origin of the fragment from which the sequence reads have been obtained; and

assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s).

5. The method of claim 4, wherein the coverage amount is in the range of 5 to 40.

6. The method of claim 4, wherein each aliquot is a well of a multi-well plate.

7. The method of claim 4, comprising amplifying the fragments before or after the aliquoting.

8. The method of claim 4, comprising amplifying the fragments or subfragments in the aliquots by polymerase chain reaction (PCR).

9. A method of determining a nucleotide sequence of one or more target polynucleotides of an organism, the method comprising the steps of:

aliquoting a coverage amount of fragments of the target polynucleotide(s) into a plurality of aliquots, wherein the coverage amount is in the range of 2 to 50, and the number of aliquots is selected to minimize the probability of having overlapping fragments within the same aliquot;

then

obtaining sequence reads of the fragments;

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

assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s);

wherein each aliquot is a well of a multi-well plate selected from a 96-well plate, a 384-well plate and a 1536-well plate.

10. A method of determining a nucleotide sequence of a target polynucleotide of an organism, the method comprising the steps of:

aliquoting a coverage amount of fragments of the target polynucleotide into a plurality of aliquots, wherein the coverage amount is in the range of 2 to 50, and the number of aliquots is selected to minimize the probability of having overlapping fragments within the same aliquot;

then

further fragmenting the fragments after the aliquoting to produce subfragments of the target polynucleotide;

obtaining sequence reads of the subfragments;

determining the respective aliquot of origin of the subfragments from which the sequence reads have been obtained; and

assembling the sequence reads to produce an assembled sequence of the target polynucleotide.

11. The method of claim 10, comprising mixing together amplicons formed from the subfragments from a plurality of the aliquots before obtaining the sequence reads.

12. A method of determining a nucleotide sequence of one or more target polynucleotides of an organism, the method comprising the steps of:

aliquoting a coverage amount of fragments of the target polynucleotide(s) into a plurality of aliquots, wherein the coverage amount is in the range of 2 to 50, and the number of aliquots is selected to minimize the probability of having overlapping fragments within the same aliquot;

amplifying the fragments in the aliquots such that each amplicon comprises a tag sequence that identifies its aliquot of origin;

then

obtaining sequence reads of the amplicons;

determining the respective aliquot of origin of the amplicons from which the sequence reads have been obtained; and

assembling the sequence reads to produce an assembled sequence of the target polynucleotide(s).

13. The method of claim 12, comprising mixing together amplicons from a plurality of the aliquots before obtaining the sequence reads.

14. A method of determining a nucleotide sequence of genomic DNA of an organism, the method comprising the steps of:

diluting the genomic DNA of the organism;

aliquoting the genomic DNA into a plurality of aliquots, wherein each aliquot comprises less than a genome equivalent of the genomic DNA;

amplifying the genomic DNA in at least some of the aliquots to produce amplified genomic DNA;

producing fragments of the amplified genomic DNA in each aliquot comprising a tag that identifies the aliquot of origin of the fragments;

mixing together the fragments from at least some of the aliquots to form a mixture;

obtaining sequence reads from fragments of the mixture;

determining the respective aliquot of origin of the sequence reads; and

assembling the sequence reads to produce an assembled genomic sequence.