Nucleic acid analysis by random mixtures of non-overlapping fragments

• US 7,901,891 B2
• Filed: 12/15/2008
• Issued: 03/08/2011
• 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:
  US12/335,168
• Publication Number:
  US 20090311691A1
• Time in Patent Office: 813 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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 nucleotide sequences of one or more target polynucleotides, said method comprising:

(a) fragmenting said one or more target polynucleotides present in a predetermined coverage amount to form a population containing overlapping first-sized fragments having an average length substantially less than those of the one or more target polynucleotides;

(b) forming a number of separate mixtures from said population of first-sized fragments, wherein said number is selected such that substantially every first-sized fragment in a separate mixture is non-overlapping with every other first-sized fragment of the same mixture and wherein the mixture of origin of each such first-sized fragment is determinable;

(c) obtaining a number of sequence reads from one or more first-sized fragments of each separate mixture;

(d) assembling said sequence reads to produce assembled sequence information comprising contigs; and

(e) providing complete or partial nucleotide sequences of said one or more target polynucleotides by ordering said assembled sequence information, wherein said ordering depends on the mixture of origin of said assembled sequence information.

2. The method of claim 1 wherein said forming step (b) comprises replicating said first-sized fragments in said separate mixtures.

3. The method of claim 1 wherein each of said sequence reads from said one or more first-sized fragments of each separate mixture has a length substantially less than those of said one or more first-sized fragments, and said number of sequence reads is selected such that sequence reads of each said separate mixture substantially covers said one or more first-sized fragments.

4. The method of claim 1, wherein said method further comprises prior to said obtaining step (c), tagging each of said first-sized fragments with an oligonucleotide tag to form tagged separate mixtures.

5. The method of claim 4, wherein prior to said obtaining step (c), said tagged separate mixtures are pooled together into a single mixture.

6. The method of claim 5 wherein said obtaining step (c) further comprises determining a nucleotide sequence of said oligonucleotide tag.

7. The method of claim 1, wherein said obtaining step (c) comprises:

(i) producing for each of said separate mixtures a plurality of target concatemers from said first-sized fragments;

(ii) forming for each of said separate mixtures a random array of target concatemers fixed to a surface at a density such that at least a majority of said target concatemers are optically resolvable; and

(iii) generating sequence reads from target concatemers of each of said separate mixtures, such sequence reads each having a length substantially less than those of said first- sized fragments, and said number of sequence reads being selected such that sequence reads of each said separate mixture substantially covers said first-sized fragments therein.

8. The method of claim 7, wherein said step wherein said generating step (iii) comprises:

(A) hybridizing one or more probes from a first set of probes to each random array under conditions that permit the formation of perfectly matched duplexes between probes from said first set and complementary sequences on said target concatemers;

(B) hybridizing one or more probes from a second set of probes to each random array under conditions that permit formation of perfectly matched duplexes between probes from said second set and complementary sequences on said target concatemers;

(C) ligating probes from said first and second sets hybridized to a target concatemer at adjacent sites to form ligated probes;

(D) identifying sequences of said ligated probes; and

(E) repeating steps (A) through (D) for each random array to generate said sequence reads.

9. The method of claim 1 wherein said one or more target polynucleotides contain repetitive sequence regions each having a length greater than those of said sequence reads.

10. The method of claim 1 wherein said ordering said assembled sequence information depends on identities of said separate mixtures from which each of said first-sized fragment is derived.

11. The method of claim 1 wherein average length of said first-sized fragments is in the range of from 50 to 2000 bases.

12. The method of claim 1 wherein average length of said first-sized fragments is in the range of from one third to one thousandth of that of said one or more target polynucleotides.

13. The method of claim 1 wherein said ordering provides haplotype information for a diploid genome.

14. A method of characterizing nucleotide sequences of one or more target polynucleotides, said method comprising:

(a) randomly fragmenting said one or more target polynucleotides present in a predetermined coverage amount to form a population containing overlapping first-sized fragments having an average length less than those of said one or more polynucleotides;

(b) forming a number of separate mixtures from said population of first-sized fragments, said number being selected such that every first-sized fragment in a separate mixture is non-overlapping with every other first-sized fragment of the same mixture;

(c) randomly fragmenting each of the first-sized fragments in each of the mixtures to form a population of second-sized fragments for each mixture having an average length less than those of the first-sized fragments and wherein the mixture of origin of each such second-sized fragment is determinable;

(d) determining sequence information from at least a portion of one or more second-sized fragments of each separate mixture; and

(e) providing complete or partial nucleotide sequences of said one or more target polynucleotides by ordering said sequence information from said separate mixtures,

wherein such ordering depends on the mixture of origin of at least a portion of said sequence information.

15. The method of claim 14 wherein said step (b) of forming comprises replicating said first-sized fragments in said separate mixtures.

16. The method of claim 14, wherein said average length of said first-sized fragments is less than 300 kilobases and said average length of said second-sized fragments is in the range of from 50 to 600 bases.

17. The method of claim 14 wherein said average length of said first-sized fragments is in the range of from one third to one thousandth of that of said one or more target polynucleotides and said average length of said second-sized fragments is in the range of from one third to one thousandth of that of said first-sized fragments.

18. The method of claim 14 wherein said predetermined coverage amount is in the range of from 3 to 30.

19. The method of claim 14 wherein said one or more target polynucleotides are one or more bacterial genomes.

20. The method of claim 14 wherein said one or more target polynucleotides are haploid strands of a diploid genome and wherein said ordering provides haplotype information for a said genome.

21. The method of claim 14 wherein said sequence information is a nucleotide sequence of said portion of said one or more second-sized fragments.

22. The method of claim 14 further comprising prior to said determining step (d), tagging each of said second-sized fragments with an oligonucleotide tag to form tagged second-sized fragments.

23. The method of claim 22, wherein said method further comprises pooling said tagged second sized fragments in said separate mixtures into a single mixture prior to said determining step (d).

24. The method of claim 23, wherein determining step (d) further comprises determining a nucleotide sequence of said oligonucleotide tag.

25. The method of claim 21, wherein said one or more target polynucleotides contain repetitive sequence regions each having a length greater than that of said nucleotide sequence.

26. The method of claim 14, wherein said method further includes prior to said forming step (b) separating said first-sized fragments by size.

27. The method of claim 14 wherein said determining step (d) comprises:

(i) forming for each of said separate mixtures a plurality of target concatemers from said second-sized fragments, each target concatemer comprising multiple copies of a second- sized fragment and each plurality of target concatemers including a number of second- sized fragments that covers said first-sized fragments of its respective separate mixture;

(ii) forming for each of said separate mixtures a random array of target concatemers fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable;

(iii) generating a number of sequence reads from target concatemers of each of said separate mixtures, such sequence reads each having a length less than those of said second-sized fragments, and each of said numbers of sequence reads selected such that sequence reads of each said separate mixture covers said second-sized fragments therein.

28. The method of claim 27 wherein generating step (iii) comprises:

(A) hybridizing one or more probes from a first set of probes to each random array under conditions that permit formation of perfectly matched duplexes between said one or more probes and complementary sequences on said target concatemers;

(B) hybridizing one or more probes from a second set of probes to each random array under conditions that permit formation of perfectly matched duplexes between one or more probes and complementary sequences on said target concatemers;

(C) ligating probes from the first and second sets hybridized to target concatemers at adjacent sites to form ligated probes;

(D) identifying sequences of said ligated probes; and

(E) repeating steps (A) through (D) for each random array until sequences of said first- sized fragments of each of said separate mixtures is determined.

29. The method of claim 14, wherein each of said first-sized fragments has an oligonucleotide tag attached to an end, said oligonucleotide tag identifying said separate mixture of said first-sized fragments.

30. A method of characterizing nucleotide sequences of one or more target polynucleotides, said method comprising the steps of:

(a) randomly fragmenting said one or more target polynucleotides present in a predetermined coverage amount to form a population containing overlapping first-sized fragments having an average length less than those of said one or more polynucleotides, said first-sized fragments being initial prior-sized fragments;

(b) generating a set of separate mixtures from said population of prior-sized fragments, such set being a tier having a number of mixtures therein, said number being selected such that every prior-sized fragment in a separate mixture is non-overlapping with every other prior-sized fragment of the same mixture;

(c) randomly fragmenting each of said prior-sized fragments in each of said separate mixtures to form a population of second-sized fragments having an average length less than that of the first-sized fragments and wherein the mixtures of origin of each second-sized fragment can be identified, said second-sized fragments being prior-sized fragments of a next step of fragmenting;

(d) repeating steps (b) and (c) until a final tier of mixtures is obtained;

(e) determining sequence information from at least a portion of one or more second-sized fragments of each mixture in said final tier; and

(f) providing complete or partial nucleotide sequences of said one or more target polynucleotides by ordering sequence information from said final tier of mixtures, wherein such ordering depends on identities of mixtures of origin of at least a portion of said sequence information.

31. A method of characterizing nucleotide sequences of one or more target polynucleotides, said method comprising:

(a) forming a plurality of tiers of mixtures that comprise a hierarchy of nested fragments of said one or more target polynucleotides, each mixture of each prior tier being divided into a number of mixtures in a subsequent tier, at least one tier having mixtures with non- overlapping fragments, and said plurality of tiers having a final tier wherein mixtures of prior tiers can be identified for each fragment of each mixture of said final tier;

(b) determining sequence information from at least a portion of one or more fragments of each mixture in said final tier; and

(c) providing complete or partial nucleotide sequences of the one or more target polynucleotides by ordering the sequence information from said final tier of mixtures, wherein such ordering depends on identity of at least one mixture of at least one tier from which a fragment is derived that gives rise to a portion of said sequence information.

32. The method of claim 31 wherein forming step (a) includes replicating said fragments of each of said mixtures of each of said subsequent tiers.

34. The method of claim 31 wherein said sequence information is a nucleotide sequence of said portion of said one or more said fragments from said final tier.

35. The method of claim 34, said method further tagging each of said fragments in each of said tiers with an oligonucleotide tag to identify each of said separate mixtures of each of said tiers.

36. The method of claim 35 wherein said step of determining said nucleotide sequence of said portion of said fragments of said final tier includes determining a nucleotide sequence of each of said oligonucleotide tags.

37. The method of claim 34 wherein said one or more target polynucleotides contain repetitive sequence regions each having a length greater than that of said nucleotide sequence.

33. The method of 31 wherein said ordering said sequence information depends on identities of said mixtures at each said tier from which each said fragment in said final tier is derived.