Sequential sequencing
View Patent ↗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.
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.
2. The method of claim 1 , wherein the at least one nucleic acid template comprises an adaptor oligonucleotide ligated to one or both ends of the at least one nucleic acid template.
3. The method of claim 2 , wherein the first sequencing primer is selectively hybridizable to a primer binding site of the adaptor oligonucleotide.
4. The method of claim 3 , wherein the second sequencing primer is not selectively hybridizable to the primer binding site of the adaptor oligonucleotide.
5. The method of claim 1 , wherein the second sequencing read comprises an informative locus.
6. The method of claim 1 , further comprising subjecting the at least one nucleic acid template to one or more additional rounds of sequencing, wherein the one or more additional rounds of sequencing comprise the steps of : (i) removing a previously annealed sequencing primer and previously generated sequencing read from the at least one nucleic acid template; (ii) annealing an additional sequencing primer to the at least one nucleic acid template; and (iii) subjecting the at least one nucleic acid template to an additional round of sequencing using the additional sequencing primer, thereby producing an additional sequencing read of the at least one nucleic acid template.
7. The method of claim 6 , wherein a first allele detected by the first sequencing read, a second allele detected by the second sequencing read, and an additional allele detected by the additional sequencing read are determined to be in phase.
8. The method of claim 6 , wherein the additional sequencing primer and the previously annealed sequencing primer are designed to hybridize to genomic locations that map to a single chromosome.
9. The method of claim 8 , wherein the genomic locations that map to a single chromosome span a distance that is less than a length of the at least one nucleic acid template.
10. The method of claim 1 , wherein the at least one nucleic acid template is a member of a sequencing library.
11. The method of claim 10 , wherein the sequencing library is a target-enriched sequencing library.
12. The method of claim 1 , wherein the first, second, and optionally additional sequencing reads comprise sequence information from a plurality of loci.
13. The method of claim 12 , wherein phasing of alleles detected at the plurality of loci is determined based upon the first, second, and optionally additional sequencing reads.
14. The method of claim 1 , wherein the at least one nucleic acid template comprises a plurality of nucleic acid templates.
15. The method of claim 1 , wherein the at least one nucleic acid template comprises at least 10,000 nucleic acid templates.
16. The method of claim 15 , wherein the at least one nucleic acid template comprises 3 billion nucleic acid templates.
17. The method of any of claim 1 , wherein the at least one nucleic acid template is coupled to a unique addressable location of the sequencing platform.
18. The method of claim 17 , comprising determining phasing of a plurality of alleles detected across two or more unique addressable locations.
19. The method of claim 1 , wherein step (d) comprises subjecting the at least one nucleic acid template to a second round of sequencing using a mixture of second sequencing primers.
20. The method of claim 19 , wherein primers within the mixture are designed and/or selected to have minimal potential to hybridize to the same nucleic acid template.