Methods and compositions for tagging and identifying polynucleotides

• US 9,194,001 B2
• Filed: 02/24/2015
• Issued: 11/24/2015
• 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:
  US14/630,566
• Publication Number:
  US 20150240302A1
• Time in Patent Office: 273 Days
• Field of Search: None
• US Class Current: 1/1
• 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 specific hybridization of the oligonucleotide tag to its tag complement on an addressable array; or such analysis can be carried out serially by successive specific hybridizations of labeled word complements, or the like.

Claims

1. A method of sequencing comprising:

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

(a) each of the cDNAs comprises;

(i) a tag that indicates from which source a cDNA is derived and (ii) separate from the tag, a binding site for a sequencing primer;

(b) the sequencing is done using the sequencing primer; and

(c) the cDNAs have been selected from a pooled cDNA sample;

to obtain, for each of the sequenced cDNAs, the nucleotide sequence of the tag associated with the cDNA and the nucleotide sequence of at least a portion of a cDNA.

2. The method of sequencing of claim 1, further comprising identifying the source of the cDNA using the sequence of the 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 the cDNAs in the pooled cDNA sample are from 50 to 10,000 different sources.

5. The method of sequencing of claim 1, wherein the tag is from 2 to 18 nucleotides in length.

6. The method of sequencing of claim 1, wherein the cDNA is in the range of 100 to 300 nucleotides in length.

7. The method of sequencing of claim 1, wherein the method further comprises analyzing the sequences to identify a sequence variation.

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

(i) pooling a plurality of samples to make a pooled sample; and

(ii) selecting tagged cDNAs from the pooled sample, wherein the tagged cDNAs correspond to one or more genes, thereby producing the plurality of cDNAs that are sequenced.

9. The method of sequencing of claim 8, wherein the selecting is done by hybridization to a probe that is immobilized to a solid support.

10. The method of sequencing of claim 8, wherein the selecting is done by hybridization to a biotinylated probe that is captured by a solid support.

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

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

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