Methods for long read sequencing
Provided herein are methods including alternating series of sequencing cycles and dark extension cycles allowing longer read lengths and addressing disadvantages of traditional nucleic acid sequencing protocols.
1. A method of extending a complementary polynucleotide hybridized to a template nucleic acid, the method comprising:
(a) contacting the complementary polynucleotide with an extension solution thereby forming an extension strand, wherein said extension solution comprises four nucleotide types, wherein only one of the four nucleotide types comprises a reversible terminator; and
(b) removing said reversible terminator and repeating step (a) for one or more cycles with said extension solution, wherein the nucleotide comprising the reversible terminator is the same nucleotide type in one or more cycles.
2. The method of claim 1 , wherein said four nucleotide types consist of: i) an adenine nucleotide, or analog thereof; ii) a thymine nucleotide, or analog thereof, or a uracil nucleotide, or analog thereof; iii) a cytosine nucleotide, or analog thereof; and iv) a guanine nucleotide, or analog thereof.
3. The method of claim 2 , wherein said adenine nucleotide, or analog thereof, comprise the reversible terminator.
4. The method of claim 2 , wherein said thymine nucleotide, or analog thereof, comprise the reversible terminator.
5. The method of claim 2 , wherein said cytosine nucleotide, or analog thereof, comprise the reversible terminator.
6. The method of claim 2 , wherein said guanine nucleotide, or analog thereof, comprise the reversible terminator.
7. The method of claim 1 , wherein prior to step a), said method comprises removing a reversible terminator from the complementary polynucleotide by contacting the complementary polynucleotide with a cleaving agent.
8. The method of claim 1 , wherein during step a), said method comprises detecting the nucleotides incorporated.
9. The method of claim 1 , wherein prior to step a), said method comprises sequencing said template nucleic acid.
10. The method of claim 1 , further comprising sequencing said template nucleic acid to generate a sequenced-extension strand.
11. The method of claim 10 , further comprising contacting the sequenced-extension strand with the extension solution; and removing said reversible terminator and repeating contacting the sequenced-extension strand with said extension solution for one or more cycles.
12. The method of claim 9 , wherein sequencing comprises incorporating one or more labeled nucleotides or labeled nucleotide analogues with a polymerase into the complementary polynucleotide, and detecting the label that identifies the one or more incorporated nucleotides.
13. The method of claim 12 , wherein 2 to 150 nucleotides are incorporated.
14. The method of claim 1 , wherein the method comprises repeating step (a) for 5 to 50 cycles.
15. The method of claim 1 , wherein the method comprises repeating step (a) for 25 to 100 cycles.
16. The method of claim 1 , wherein the template nucleic acid is immobilized to a solid support.
17. The method of claim 1 , wherein the template nucleic acid is at least 1000 bases (1kb), at least 2 kb, at least 4 kb, at least 6 kb, at least 10 kb, at least 20 kb, at least 30 kb, at least 40 kb, or at least 50 kb in length.
18. The method of claim 10 , wherein the method produces one or more sequencing reads comprising joined discontinuous nucleic acid sequences collectively spanning a length of about 100 to about 5000 bases of the template nucleic acid.
19. The method of claim 18 , further comprising aligning the one or more sequencing reads to a reference sequence.
20. The method of claim 19 , further comprising generating a consensus sequence from the aligning of one or more sequencing reads, wherein the consensus sequence comprises (i) a nucleic acid sequence in one or more first sequencing reads that is absent from one or more second sequencing reads, and (ii) a nucleic acid sequence in one or more of the second sequencing reads that is absent from the one or more first sequencing reads.
21. The method of claim 1 , wherein the template nucleic acid is an RNA transcript.
22. The method of claim 1 , wherein the template nucleic acid comprises a gene or a gene fragment, wherein the gene or gene fragment is a cancer-associated gene or fragment thereof, T cell receptor (TCR) gene or fragment thereof, or a B cell receptor (BCR) gene, or fragment thereof.
23. The method of claim 10 , wherein sequencing comprises incorporating one or more labeled nucleotides or labeled nucleotide analogues with a polymerase into the complementary polynucleotide, and detecting the label that identifies the one or more incorporated nucleotides.
24. The method of claim 1 , wherein the template nucleic acid is within a sample comprising cells or tissues.
25. A method of extending a polynucleotide hybridized to a template nucleic acid, the method comprising:
(a) contacting the polynucleotide with a first extension solution thereby forming an extension strand, wherein said first extension solution comprises a plurality of adenine nucleotides, a plurality of thymine nucleotides, a plurality of cytosine nucleotides, and a plurality of guanine nucleotides, wherein the nucleotides of one plurality comprise a reversible terminator;
(b) removing said reversible terminator; and
(c) contacting the polynucleotide with the same extension solution of step (a).
26. The method of claim 25 , wherein said template nucleic acid is in a cell or tissue.
27. The method of claim 25 , further comprising sequencing said template nucleic acid to generate a sequenced-extension strand.
28. The method of claim 25 , wherein steps (a), (b), and (c) comprise a cycle, the method further comprises repeating steps (a), (b), and (c) for 5 to 500 cycles.
29. The method of claim 25 , wherein the adenine nucleotides comprise a reversible terminator or the thymine nucleotides comprise a reversible terminator.
30. The method of claim 25 , wherein the cytosine nucleotides comprise a reversible terminator or the guanine nucleotides comprise a reversible terminator.
31. The method of claim 25 , wherein said template nucleic acid is on a cell.
32. The method of claim 1 , wherein the reversible terminator is comprises an azido moiety, a disulfide moiety, or an allyl moiety.
33. The method of claim 1 , wherein the reversible terminator comprises the formula:
34. The method of claim 1 , wherein the reversible terminator is comprises an azido moiety, a disulfide moiety, or an allyl moiety.
35. The method of claim 1 , wherein the reversible terminator comprises the formula: