Sequential sequencing
First Claim
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1. A method, comprising:
- a) coupling at least one nucleic acid template to a sequencing platform;
b) subjecting the at least one nucleic acid template to a first round of sequencing using a first sequencing primer, wherein the first sequencing primer comprises a 3′
terminal nucleotide which anneals to a first binding site in the at least one nucleic acid template, wherein the first round of sequencing produces a first sequencing read of the at least one nucleic acid template;
c) removing the first annealed sequencing primer and the first sequencing read from the at least one nucleic acid template; and
d) subjecting the at least one nucleic acid template to a second round of sequencing using a second sequencing primer, wherein the second sequencing primer comprises a 3′
terminal nucleotide which anneals to a second binding site in the at least one nucleic acid template which is more than 5 nt away from the first binding site, wherein the second round of sequencing produces a second sequencing read of the at least one nucleic acid template.
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Abstract
Described herein are improved methods, compositions and kits for next generation sequencing (NGS). The methods, compositions and kits described herein enable phasing of two or more nucleic acid sequences in a sample, i.e. determining whether the nucleic acid sequences (which can comprise regions of sequence variation) are located on the same chromosome and/or the same chromosomal fragment. Phasing information can be obtained by performing multiple, successive sequencing reactions from the same immobilized nucleic acid template. The methods, compositions and kits provided herein can be useful, for example, for haplotyping, SNP phasing, or for determining downstream exons in RNA-seq.
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Citations
20 Claims
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1. A method, comprising:
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a) coupling at least one nucleic acid template to a sequencing platform; b) subjecting the at least one nucleic acid template to a first round of sequencing using a first sequencing primer, wherein the first sequencing primer comprises a 3′
terminal nucleotide which anneals to a first binding site in the at least one nucleic acid template, wherein the first round of sequencing produces a first sequencing read of the at least one nucleic acid template;c) removing the first annealed sequencing primer and the first sequencing read from the at least one nucleic acid template; and d) subjecting the at least one nucleic acid template to a second round of sequencing using a second sequencing primer, wherein the second sequencing primer comprises a 3′
terminal nucleotide which anneals to a second binding site in the at least one nucleic acid template which is more than 5 nt away from the first binding site, wherein the second round of sequencing produces a second sequencing read of the at least one nucleic acid template. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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Specification