Methods and compositions for tagging and identifying polynucleotides
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 submixtues 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.
1. A method of identifying a polynucleotide selected from a mixture of tagged polynucleotides, the method comprising the steps of:
selecting one or more tagged polynucleotides having a particular nucleic acid sequence at one or more loci from a mixture of tagged polynucleotides, said mixture comprising polynucleotides from a plurality of different polynucleotide samples wherein the polynucleotides in each of said different polynucleotide samples are tagged with an oligonucleotide tag unique to the sample and comprising one or more words; and
determining the nucleotide sequence of: a) at least part of a selected polynucleotide and b) at least one word of the unique oligonucleotide tag on said selected polynucleotide to identify from which of said plurality of different polynucleotide samples said selected polynucleotide is derived.
2. The method of claim 1 , wherein said selecting step selects tagged polynucleotides from said mixture of tagged polynucleotides having a genetic variation at said one or more loci.
3. The method of claim 1 , wherein said one or more loci are polymorphic loci.
4. The method of claim 1 , wherein said selecting is based on one or more of: specific hybridization and differential duplex stability, template-driven ligation, template-driven strand extension, and exonuclease digestion.
5. The method of claim 1 , wherein said determining step comprises determining the nucleotide sequence of at least one word of the unique oligonucleotide tag on said selected one or more tagged polynucleotides in successive steps.
6. The method of claim 5 , wherein each of said successive steps comprises contacting said selected one or more tagged polynucleotides under hybridizing conditions to a word-specific probe.
7. The method of claim 1 wherein each of said unique oligonucleotide tags comprises multiple words, said multiple words having an order, and wherein said determining step comprises determining the order of said words.
8. The method of claim 1 , wherein said determining step comprises determining the nucleotide sequence of at least one word of the unique oligonucleotide tag on said selected one or more tagged polynucleotides simultaneously.
9. The method of claim 8 , wherein said determining step comprises contacting said selected one or more tagged polynucleotides under hybridizing conditions to a solid phase support comprising complements to said unique oligonucleotide tags.
10. The method of claim 9 , wherein said solid phase supports is an addressable array.
11. The method of claim 1 , wherein said plurality of different polynucleotide samples are a plurality of different genomic samples.
12. The method of claim 1 , wherein said polynucleotides in each of said plurality of different polynucleotide samples are polynucleotide fragments.
13. The method of claim 1 , wherein each of said unique polynucleotide tags comprises from four to eight words.
14. The method of claim 1 , wherein each of said words comprises from 2 to 8 nucleotides.
15. The method of claim 1 , wherein each of said unique oligonucleotide tags is a ligation tag.
16. The method of claim 1 , further comprising producing said mixture of tagged polynucleotides, said producing step comprising:
(a) providing:
(i) a mutually discriminable set containing a plurality of different words; and
(ii) said plurality of different polynucleotide samples;
(b) attaching one or more of said different words to the polynucleotides in each of said plurality of different polynucleotide samples to form tag-polynucleotide conjugates , wherein the polynucleotides in each of said different polynucleotide samples are tagged with a unique oligonucleotide tag comprising one or more words; and
(c) combining said tag-polynucleotide conjugates to produce said mixture of tagged polynucleotides.
17. The method of claim 1 , wherein said plurality of different polynucleotide samples are a plurality of different cDNA samples.
18. The method of claim 17 , wherein polynucleotides having a particular nucleic acid sequence at one or more loci are retrieved in the retrieving step.
19. The method of claim 1 , wherein said determining step comprises determining the nucleotide sequence of at least one word of the unique oligonucleotide tag on said selected one or more tagged polynucleotides.
20. A method of identifying a polynucleotide selected from a mixture of tagged polynucleotides, the method comprising the steps of:
retrieving one or more tagged polynucleotides from a mixture of tagged polynucleotides, said mixture comprising polynucleotides from a plurality of different polynucleotide samples wherein the polynucleotides in each of said different polynucleotide samples are tagged with an oligonucleotide tag unique to the sample and comprising one or more words; and
determining the nucleotide sequence of: a) at least part of a retrieved polynucleotide and b) at least one word of the unique oligonucleotide tag on said retrieved polynucleotide to identify from which of said plurality of different polynucleotide samples said retrieved polynucleotides is derived.