Nucleic acid sequencing and process
The present invention is directed to compositions and methods for nucleic acid identification and detection. Compositions and methods of the present invention include extracting and fragmenting target nucleic acids from a sample, using the fragmented target nucleic acids to produce target nucleic acid templates and subjecting those target nucleic acid templates to amplification methods to form nucleic acid nanoballs. The invention also includes methods of detecting and identifying sequences using various sequencing applications, including sequencing by ligation methods.
1. A method of determining a first nucleotide at a detection position of a target sequence comprising a plurality of detection positions, said method comprising:
a) providing an array comprising a plurality of concatemers randomly disposed on a surface, wherein each concatemer comprises a plurality of monomers and each monomer comprises:
i) a target domain of said target sequence comprising a set of target detection positions;
ii) an adaptor comprising a first anchor site;
b) hybridizing a first anchor probe to said first anchor site;
c) hybridizing a fully degenerate second anchor probe to the target sequence, adjacent to said first anchor site, or,
hybridizing a partially degenerate second anchor probe to a second anchor site in the adaptor adjacent to the first anchor site, wherein said partially degenerate second anchor probe also hybridizes to the target sequence;
d) hybridizing a sequencing probe to said target domain, wherein said sequencing probe comprises:
i) a first probe domain complementary to said target domain;
ii) a unique nucleotide at a first interrogation position; and
iii) a label;
under conditions wherein if said unique nucleotide is complementary to said first nucleotide, said sequencing probe hybridizes to said target domain;
e) ligating said anchor probes and said sequencing probe; and
f) identifying said first nucleotide
wherein said ligating step (e) is driven to between about 20 percent and about 90 percent completion.
2. A method according to claim 1 wherein a set of sequencing probes are contacted with said surface, each sequencing probe comprising:
a) a first probe domain complementary to said target domain;
b) a unique nucleotide at a first interrogation position; and
c) a label;
wherein each label of said set corresponds to said unique nucleotide.
3. A method according to claim 1 wherein each of said monomers comprises a plurality of adaptors.
4. A method according to claim 3 wherein at least one of said adaptors comprises at least one Type IIs endonuclease recognition site.
5. A method according to claim 1 wherein steps b) e) are repeated to identify a second nucleotide at a second detection position.
6. A method according to claim 1 , wherein said second anchor probe comprises a set of second anchor probes comprising at least three degenerate bases that hybridizes to said sequences outside said second anchor site.
7. A method according to claim 1 , wherein said second anchor probe comprises at least one terminus that is selectively activatable for ligation.
8. A method according to claim 1 wherein said surface is functionalized.
9. A method according to claim 8 wherein said functionalized surface comprises functional moieties selected from the group consisting of amines, silanes, and hydroxyls.
10. A method according to claim 1 wherein said surface comprises a plurality of spatially distinct regions comprising said immobilized concatemers.
11. A method according to claim 1 wherein said concatemers are immobilized on said surface using capture probes.
12. A method according to claim 1 further comprising fragmenting genomic nucleic acid to form target sequences.
13. A method according to claim 1 wherein said target sequence is a genomic nucleic acid sequence.
14. A method according to claim 13 wherein said genomic nucleic acid sequences are human.
15. A method according to claim 1 wherein the second anchor probe comprises from 5 to 9 bases.
16. A method according to claim 1 wherein the second anchor probe is a six-mer.
17. A method according to claim 1 wherein the said ligating step (e) is driven to between about 20 percent completion.
18. A method according to claim 1 wherein the said ligating step (e) is driven to between about 25 percent completion.
19. A method according to claim 1 wherein the said ligating step (e) is driven to between about 30 percent completion.
20. A method according to claim 1 wherein the said ligating step (e) is driven to between about 35 percent completion.
21. A method according to claim 1 wherein the said ligating step (e) is driven to between about 40 percent completion to about 85 percent completion.