Methods and compositions for long fragment read sequencing

• US 8,592,150 B2
• Filed: 06/15/2010
• Issued: 11/26/2013
• Est. Priority Date: 12/05/2007
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje, Peters, Brock A., Alexeev, Andrei, Hong, Peter
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US12/816,365
• Publication Number:
  US 20110033854A1
• Time in Patent Office: 1,260 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components

Abstract

The present invention is directed to methods and compositions for long fragment read sequencing. The present invention encompasses methods and compositions for preparing long fragments of genomic DNA, for processing genomic DNA for long fragment read sequencing methods, as well as software and algorithms for processing and analyzing sequence data.

Claims

1. A method of obtaining sequence information from a genome, said method comprising:

(a) providing a population of first fragments of said genome;

(b) preparing emulsion droplets of said first fragments, such that each emulsion droplet comprises a subset of said population of first fragments;

(c) fragmenting said first fragments, thereby obtaining a population of second fragments within each emulsion droplet, such that said second fragments are shorter than said first fragments;

(d) combining individual emulsion droplets comprising said second fragments with individual emulsion droplets comprising adaptor tags or adaptor tag combinations, thereby forming fused droplets;

(e) ligating said second fragments with said adaptor tags or adaptor tag combinations within the fused droplets to form tagged fragments;

(f) combining the fused droplets to produce a mixture containing tagged fragments;

(g) obtaining sequence reads from tagged fragments in the mixture;

(h) assembling the sequence reads to produce assembled sequence information for the genome, wherein the assembled sequence information comprises heterozygous loci; and

(i) phasing the heterozygous loci using sequence information from the adaptor tags.

2. The method of claim 1, wherein said emulsion droplets of said adaptor tags each individually comprises at least two sets of different tag components such that fragments in at least some of said emulsion droplets are tagged with different combinations of said tag components in said ligating step (e).

3. The method of claim 2, wherein at least 1000 of said emulsion droplets in said ligating step (e) comprise fragments tagged with different combinations of said tag components.

4. The method of claim 3, wherein at least 10,000, 30,000, or 100,000 of said emulsion droplets in said ligating step (e) comprise fragments tagged with different adaptor tags or adaptor tag combinations.

5. The method of claim 2, wherein said tag components are from a set of over 1000 distinct barcodes prepared as a population of liquid drops in oil.

6. The method of claim 1, wherein said emulsion droplets of said first fragments comprise only 1-5 first fragments in each droplet.

7. The method of claim 1, wherein said emulsion droplets of said second fragments or said emulsion droplets of said adaptors further comprise ligase.

8. The method of claim 1, wherein each adaptor tag or adaptor tag combination comprises a segment that is common to all adaptor tags and a segment comprising an identifier sequence.

9. The method of claim 1, wherein each adaptor tag is blocked at one end to control orientation of the tag when ligated to a genome fragment.

10. The method of claim 1, wherein step (c) comprises amplifying the first fragments so as to replace nucleotides therein with the nucleotide analogs, and then either excising the nucleotide analogs or producing a nick either immediately 3′

or 5′

to the nucleotide analogs, thereby forming gapped nucleic acids.

11. The method of claim 1, wherein the adaptor tag or adaptor tag combination comprises eight bases or a combination of 2×

5 bases.

12. The method of claim 1, wherein the sequence reads in step (g) are obtained by a process comprising probe anchor ligation.

13. A method of obtaining sequence information from a genome, said method comprising:

(a) providing a population of first fragments of said genome;

(b) preparing emulsion droplets of said first fragments, such that each emulsion droplet comprises a subset of said population of first fragments;

(c) fragmenting said first fragments, thereby obtaining a population of second fragments within each emulsion droplet, such that said second fragments are shorter than said first fragments;

(d) combining individual emulsion droplets comprising said second fragments with individual emulsion droplets comprising adaptor tags or adaptor tag combinations, thereby forming fused droplets;

(e) ligating said second fragments with said adaptor tags or adaptor tag combinations within the fused droplets to form tagged fragments;

(f) combining the fused droplets to produce a mixture containing tagged fragments;

(g) obtaining sequence reads from tagged fragments in the mixture,

(h) combining sequence reads from tagged fragments having the same adaptor tags to produce sequences of longer contiguous regions; and

(i) assembling the sequence reads into sequence information for the genome wherein the sequence information comprises said sequences of longer contiguous regions.

14. The method of claim 13, wherein said emulsion droplets of said adaptor tags each individually comprises at least two sets of different tag components such that fragments in at least some of said emulsion droplets are tagged with different combinations of said tag components in said ligating step (e).

15. The method of claim 13, wherein said emulsion droplets of said second fragments or said emulsion droplets of said adaptors further comprise ligase.

16. The method of claim 13, wherein each adaptor tag or adaptor tag combination comprises a segment that is common to all adaptor tags and a segment comprising an identifier sequence.

17. The method of claim 13, wherein each adaptor tag is blocked at one end to control orientation of the tag when ligated to a genome fragment.

18. The method of claim 13, wherein step (c) comprises amplifying the first fragments so as to replace nucleotides therein with the nucleotide analogs, and then either excising the nucleotide analogs or producing a nick either immediately 3′

or 5′

to the nucleotide analogs, thereby forming gapped nucleic acids.

19. The method of claim 13, wherein the adaptor tag or adaptor tag combination comprises eight bases or a combination of 2×

5 bases.

20. The method of claim 13, wherein the sequence reads in step (g) are obtained by a process comprising probe anchor ligation.

21. The method of claim 13, wherein step (h) generates sequence data equivalent to sequencing single DNA molecules of greater than 100 kb.

22. The method of claim 13, which is a method for sequencing a complete diploid genome.

23. The method of claim 13, which is a method for haplotyping a diploid chromosome.