Nucleic Acid Analysis by Random Mixtures of Non-Overlapping Fragments

• US 20110319281A1
• Filed: 01/31/2011
• Published: 12/29/2011
• 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
• Granted Patent: US 8,765,379 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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, the method comprising the steps of:

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

forming a number of separate mixtures from the population of first-sized fragments, such number being 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 such that the mixture of origin of each such first-sized fragment is determinable;

determining sequence information from at least a portion of one or more first-sized fragments of each mixture; and

providing complete or partial nucleotide sequences of the one or more target polynucleotides by ordering the sequence information from the separate mixtures, wherein such ordering depends on the mixture of origin of at least a portion of the sequence information.

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

3. The method of claim 2 wherein said step of determining includes generating for each of said separate mixtures sequence reads from a number of said first-sized fragments, the sequence reads each having a length substantially less than those of said first-sized fragments, and each of such numbers being selected such that the sequence reads of each said separate mixture substantially covers said first-sized fragments therein.

6. The method of claim 3 wherein said step of determining includes:

generating for each of said separate mixtures a plurality of target concatemers from said first-sized fragments, each target concatemer comprising multiple copies of a portion of a first-sized fragment and each plurality of target concatemers including a number of such portions that substantially covers said first-sized fragment;

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; and

generating a number of 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 each of such numbers of sequence reads being selected such that the sequences reads of each said separate mixture substantially covers said first-sized fragments therein.

7. The method of claim 6 wherein said step of generating said number of sequence reads 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 the one or more probes and complementary sequences on target concatemers;

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

(c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites;

(d) identifying the sequences of the ligated first and second probes; and

(e) repeating steps (a) through (d) for each random array until the sequences of said first-sized fragments of each of said separate mixtures is substantially determined from the identities of the sequences of the ligated probes.

4-5. -5. (canceled)

8-12. -12. (canceled)

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

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

forming a number of separate mixtures from the population of first-sized fragments, such number being 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;

fragmenting each of the first-sized fragments in each of the mixtures to form a population of second-sized fragments for each mixture such that each second-sized fragment has an average length substantially less than those of the first-sized fragments and such that the mixture of origin of each such second-sized fragment is determinable;

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

providing complete or partial nucleotide sequences of the one or more target polynucleotides by ordering the sequence information from the aliquots, wherein such ordering depends on the mixture of origin of at least a portion of the sequence information.

14. The method of claim 13 wherein said step of forming includes replicating said first-sized fragments in said separate mixtures.

25. The method of claim 14 wherein said step of determining said nucleotide sequence of each of said second-sized fragments comprises the steps of:

generating 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 substantially covers said first-sized fragments of its respective separate mixture;

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;

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

26. The method of claim 25 wherein said step of generating said number of sequence reads 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 the one or more probes and complementary sequences on target concatemers;

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

(c) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites;

(d) identifying the sequences of the ligated first and second probes; and

(e) repeating steps (a) through (d) for each random array until the sequences of said first-sized fragments of each of said separate mixtures is substantially determined from the identities of the sequences of the ligated probes.

15-24. -24. (canceled)

27-28. -28. (canceled)

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

forming a plurality of tiers of mixtures that comprise a hierarchy of nested fragments of the 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 substantially nonoverlapping fragments, and the plurality of tiers having a final tier wherein mixtures of prior tiers can be identified for each fragment of each mixture of the final tier;

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

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

30. The method of claim 29 wherein said step of forming includes replicating said fragments of each of said mixtures of each of said subsequent tiers.

32. The method of claim 30 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.

33. The method of claim 30 wherein said predetermined coverage amount is in the range of from 5 to 30 and wherein said second-sized fragments are non-overlapping in at least sixty-percent of said separate mixtures.

34. The method of claim 30 wherein said sequence information is a nucleotide sequence of said portion of said one or more said fragments from said final tier, each nucleotide sequence having a length.

35. The method of claim 34 further including a step of 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 30 wherein said one or more target polynucleotides contain repetitive sequence regions each having a length greater than that of said nucleotide sequence.

31. The method of 30 wherein said ordering said sequence information depends on the identities of said mixtures at each said tier from which each said fragment in said final tier is derived.