Tagged fragment library configured for genome or cDNA sequence analysis

• US 9,637,785 B2
• Filed: 08/20/2013
• Issued: 05/02/2017
• Est. Priority Date: 06/15/2005
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/971,806
• Publication Number:
  US 20140011688A1
• Time in Patent Office: 1,351 Days
• Field of Search: None
• 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 DNA library configured for nucleotide sequencing of a genome, the library comprising a plurality of constructs wherein:

each construct contains a fragment of the genome tagged with an oligonucleotide;

at least 96 of the constructs contain different genome fragments joined to oligonucleotide tags having different sequences;

some of the constructs contain different genome fragments joined to oligonucleotide tags having the same sequence; and

constructs that contain oligonucleotide tags having the same sequence more often contain fragment sequences that occur within 100 kb of each other in the genome, compared with constructs in the library that contain oligonucleotide tags having different sequences.

2. The DNA library of claim 1, wherein some of the fragments containing oligonucleotide tags having different sequences are aliquoted into separate aliquots, and fragments containing oligonucleotide tags having the same sequences are contained in the same aliquot.

3. The DNA library of claim 1, wherein the constructs are combined in a single mixture.

4. The DNA library of claim 1, wherein the constructs further contain one or more adaptor sequences that are common to most of the constructs.

5. The DNA library of claim 1, wherein the constructs are configured so that sequence reads of no more than 1,000 nucleotides can be obtained that include the sequence of the respective oligonucleotide tag and at least part of the sequence of the respective fragment.

6. The DNA library of claim 1, wherein the median length of the fragments is 30 to 3,000 bases.

7. The DNA library of claim 1, wherein the probability of constructs in the library containing fragments of the genome having overlapping sequences joined to oligonucleotide tags having the same sequence is less than 1%.

8. The DNA library of claim 1, wherein the constructs contain two fragments that have sequences that are nearby but not immediately beside each other in the genome separated by a common adaptor sequence.

9. The DNA library of claim 1, wherein all the constructs together contain at least a majority of the nucleotide sequence of the genome.

10. The DNA library of claim 1, containing genomes of multiple organisms.

11. A library configured for nucleotide sequencing of a target polynucleotide, the library comprising a plurality of nucleic acid constructs wherein:

each construct contains a fragment of the target polynucleotide tagged with an oligonucleotide;

at least 96 of the constructs contain different fragments of the target polynucleotide joined to oligonucleotide tags having different sequences;

some of the constructs contain different fragments of the target polynucleotide joined to oligonucleotide tags having the same sequence; and

constructs that contain oligonucleotide tags having the same sequence more often contain fragments that occur within 100 kb of each other in the target polynucleotide, compared with constructs in the library that contain oligonucleotide tags having different sequences.

12. The library of claim 11, wherein the target polynucleotide comprises a mammalian genome.

13. The library of claim 11, wherein the target polynucleotide comprises cDNA prepared from a biological sample.

14. A cDNA library configured for characterizing gene expression in a biological sample, the library comprising a plurality of constructs wherein:

each construct contains at least a fragment of a cDNA sequence obtained by reverse transcribing an mRNA transcript in the biological sample joined to an oligonucleotide tag;

at least 96 of the constructs contain different cDNA sequences tagged with oligonucleotides having different sequences;

some of the constructs contain different cDNA sequences tagged with oligonucleotides having the same sequence; and

constructs that contain oligonucleotide tags having the same sequence more often contain sequences from an mRNA transcript of the same gene, compared with constructs in the library that contain oligonucleotide tags having different sequences.

15. The cDNA library of claim 14, wherein the constructs are configured so that sequence reads of no more than 1,000 nucleotides can be obtained that include the sequence of the respective oligonucleotide tag and at least part of the sequence of the respective fragment.

16. The cDNA library of claim 14, wherein the median length of the fragments is 30 to 3,000 bases.

17. The cDNA library of claim 14, wherein the probability of constructs in the library containing fragments of the cDNA having overlapping sequences joined to oligonucleotide tags having the same sequence is less than 1%.

18. The cDNA library of claim 14, wherein the constructs are mate pairs containing two fragments that have sequences that are nearby but not immediately beside each other in the cDNA separated by a common adaptor sequence.

19. The cDNA library of claim 14, wherein some of the fragments containing oligonucleotide tags having different sequences are aliquoted into separate aliquots and fragments containing oligonucleotide tags having the same sequences are contained in a common aliquot.

20. The cDNA library of claim 14, wherein the constructs are combined in a single mixture.