Sequential sequencing

• US 10,619,206 B2
• Filed: 11/10/2017
• Issued: 04/14/2020
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Tecan Genomics
• Original Assignee: Tecan Genomics
• Inventors: Amorese, Doug, Schroeder, Benjamin G., Scolnick, Jonathan
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/809,638
• Publication Number:
  US 20180112264A1
• Time in Patent Office: 886 Days
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 Sequence alignment; Homology search

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.

Claims

1. A method, comprising:

coupling a nucleic acid template strand to a solid support in a sequencing platform;

sequencing the template strand using a first primer to produce a first sequencing read, the first primer comprising a 3′

portion that anneals to a first binding site in the template strand;

removing the first primer and an extension product from the template strand;

annealing a second primer to a genomic location in the template strand that is more than 5 nucleotides away from a binding site of the first primer, wherein a 3′

portion of the second primer is complementary to the genomic location in the template strand; and

sequencing the template strand using the second primer to produce a second sequencing read, wherein at least one of the sequencing steps comprises sequencing by synthesis.

2. The method of claim 1, wherein the sequencing comprises sequencing by synthesis using a semiconductor chip.

3. The method of claim 2, wherein the semiconductor chip comprises an ion-sensitive field effect transistor and a micro-machined well.

4. The method of claim 2, wherein the at least one step of sequencing by synthesis comprises sensing protons.

5. The method of claim 1, wherein the second binding site is more than 150 nt away from the first binding site.

6. The method of claim 2, wherein the coupling comprises conjugating the template strand to the solid support of the sequencing platform.

7. The method of claim 6, wherein the solid support comprises a bead.

8. The method of claim 1, wherein an adaptor oligonucleotide is ligated to one or both ends of the template strand.

9. The method of claim 8, wherein the first binding site is within the adaptor oligonucleotide.

10. The method of claim 9, wherein the second sequencing primer is not complementary to the adaptor oligonucleotide.

11. The method of claim 1, wherein the second sequencing read comprise an informative locus.

12. The method of claim 11, wherein the informative locus is a polymorphic locus.

13. The method of claim 1, wherein the first sequencing read comprises a first informative locus and wherein the second sequencing read comprises a second informative locus.

14. The method of claim 13, comprising determining that a first allele detected at the first informative locus and a second allele detected at the second informative locus are in phase.

15. The method of claim 14, wherein the first allele and the second allele are separated by a distance of over 150 bases.

16. The method of claim 1, further comprising subjecting the template strand to an additional round of sequencing by:

removing a previously annealed primer from the template strand;

annealing an additional primer to a second genomic location in the template strand; and

sequencing the template strand from the additional primer, thereby producing an additional sequencing read.

17. The method of claim 16, 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.