Methods and compositions for tagging and identifying polynucleotides

• US 8,318,433 B2
• Filed: 03/20/2012
• Issued: 11/27/2012
• Est. Priority Date: 02/10/2005
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/425,215
• Publication Number:
  US 20120277107A1
• Time in Patent Office: 252 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• 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: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention provides methods and compositions for attaching oligonucleotide tags to polynucleotides for the purpose of carrying out analytical assays in parallel and for decoding the oligonucleotide tags of polynucleotides selected in such assays. Words, or subunits, of oligonucleotide tags index submixtures in successively more complex sets of submixtures (referred to herein as “tiers” of submixtures) that a polynucleotide goes through while successive words are added to a growing tag. By identifying each word of an oligonucleotide tag, a series of submixtures is identified including the first submixture that contains only a single polynucleotide, thereby providing the identity of the selected polynucleotide. The analysis of the words of an oligonucleotide tag can be carried out in parallel, e.g. by hybridization of the oligonucleotide tag to its tag complement on an addressable array; or such analysis can be carried out serially by successive hybridizations of labeled word complements.

Claims

1. A method of sequencing comprising:

sequencing a plurality of genomic fragments, wherein the genomic fragments are obtained from a plurality of different sources, and wherein;

i. each of said genomic fragments comprises an oligonucleotide tag that comprises a unique nucleotide sequence that indicates from which source a genomic fragment is derived; and

, separate from said unique nucleotide sequence, a binding site for a sequencing primer;

ii. said sequencing of the plurality of genomic fragments is done using said sequencing primer; and

iii. said genomic fragments correspond to one or more selected portions of a genome that have been isolated from a pooled genomic sample by specific hybridization;

to obtain, for each of the sequenced fragments, the unique nucleotide sequence of an oligonucleotide tag and the nucleotide sequence of at least a portion of a genomic fragment.

2. The method of sequencing of claim 1, further comprising identifying the source of the nucleotide sequence using the unique sequence of the oligonucleotide tag.

3. The method of sequencing of claim 1, wherein the different sources are different human individuals.

4. The method of sequencing of claim 1, wherein said genomic fragments contain a nucleotide sequence that varies in said different sources.

5. The method of sequencing of claim 1, wherein said plurality of different sources comprise 50 to 10,000 different sources.

6. The method of sequencing of claim 1, wherein the unique nucleotide of said oligonucleotide tag is from 2 to 18 nucleotides in length.

7. The method of sequencing of claim 1, wherein said genomic fragments are fragments in the range of 100 to 300 nucleotides in length.

8. The method of sequencing of claim 1, wherein the plurality of genomic fragments are made by the following process:

a) separately ligating an oligonucleotide tag to each of a plurality of samples that comprise fragmented genomic DNA to produce a plurality of adaptor-ligated samples, wherein;

i. each of said samples is made from a different source;

ii. each of said samples is ligated to a different oligonucleotide tag;

iii. each oligonucleotide tag comprises at least one word comprising a unique nucleotide sequence that distinguishes said oligonucleotide tag from other oligonucleotide tags; and

iv. each adaptor-ligated sample comprises adaptor-ligated genomic fragments that contain an adaptor and a genomic fragment;

b) pooling said plurality of adaptor-ligated samples to make a pooled sample;

c) isolating from said pooled sample adaptor-ligated genomic fragments that correspond to one or more genomic loci, thereby producing the plurality of genomic fragments.

9. The method of sequencing of claim 8, wherein said isolating is done by hybridizing said pooled sample to a probe that is immobilized to a solid support.

10. The method of sequencing of claim 8, wherein said isolating is done by hybridizing said pooled sample to a biotinylated probe that is captured by a solid support.

11. The method of sequencing of claim 8, wherein said isolating is done by exonuclease digestion of non-circularized probes.

12. The method of sequencing of claim 8, wherein said isolating is done by template-driven ligation.

13. The method of sequencing of claim 8, wherein said isolating is done by template-driven strand extension.