Methods for indexing samples and sequencing multiple polynucleotide templates

• US 10,457,985 B2
• Filed: 11/30/2016
• Issued: 10/29/2019
• Est. Priority Date: 02/02/2007
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Bignell, Helen, Fraser, Louise, Gormley, Niall Anthony
• Primary Examiner(s): Kim, Young J
• Application Number:
  US15/365,266
• Publication Number:
  US 20170145499A1
• Time in Patent Office: 1,063 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

The invention relates to methods for indexing samples during the sequencing of polynucleotide templates, resulting in the attachment of tags specific to the source of each nucleic acid sample such that after a sequencing run, both the source and sequence of each polynucleotide can be determined. Thus, the present invention pertains to analysis of complex genomes (e.g., human genomes), as well as multiplexing less complex genomes, such as those of bacteria, viruses, mitochondria, and the like.

Claims

1. A method for sequencing nucleic acid sequences and identifying subsets of nucleic acid sequences, each subset of nucleic acid sequences isolated from a different source, the method comprising the steps of:

(a) providing at least two different samples of randomly fragmented double stranded nucleic acid targets, wherein each of the randomly fragmented double stranded nucleic acid targets is isolated from a different source;

(b) ligating a sample specific tagged adaptor to each end of each target fragment of each sample to generate adaptor-target-adaptors, wherein the tagged adaptor comprises at least one region of single stranded nucleic acid and a region of double stranded nucleic acid comprising a sample specific tag sequence that differentiates adaptor-target-adaptors originating from different samples, and wherein the sample specific tag sequence is attached directly to the target fragment in each adaptor-target-adaptor with no intervening nucleotides;

(c) amplifying the adaptor-target-adaptors of each sample with a pair of amplification primers complementary to the sample specific tag sequence to generate amplified adaptor-target-adaptors from each sample;

(d) pooling the amplified adaptor-target-adaptors of the at least two different samples;

(e) immobilizing the pooled adaptor-target-adaptors on a surface;

(f) sequencing the immobilized adaptor-target-adaptors on the surface to determine a sequence read of each immobilised adaptor-target-adaptor and identify the sample specific tag sequence of each immobilised adaptor-target-adaptor, thereby determining nucleic acid sequences of the immobilized adaptor-target-adaptors and identifying each of the immobilized adaptor-target-adaptors as a member of a subset of nucleic acid sequences.

2. The method of claim 1, wherein the sequencing of the target fragment and the sample specific tag sequence is performed in a single sequencing read.

3. The method of claim 1, wherein the sequencing of the target fragment and the sample specific tag sequence is performed in two sequencing reads.

4. The method of claim 3, wherein the solid surface is a flow cell or a collection of beads.

5. The method of claim 4, wherein the beads are isolated as single beads in an emulsion.

6. The method of claim 1, wherein the immobilized adaptor-target-adaptors are amplified on the solid surface prior to sequencing to generate an amplified array.

7. The method of claim 6, wherein the amplified array is a clustered array of amplified single molecules.

8. The method of claim 7, wherein the clustered array is formed by solid-phase nucleic acid amplification.

9. The method of claim 8, wherein the clustered array is formed by isothermal solid-phase nucleic acid amplification.

10. The method of claim 1, wherein the sequencing step comprises cycles of ligation with labelled oligonucleotides.

11. The method of claim 10, wherein the oligonucleotides are labelled with fluorophores.

12. The method of claim 1, wherein the sequencing comprises cycles of addition of nucleotides.

13. The method of claim 12, wherein the nucleotides are labelled.

14. The method of claim 13, wherein the nucleotides are labelled with fluorophores.

15. The method of claim 1, wherein the sequencing step is carried out on a flow cell or an array of immobilised beads.

16. The method of claim 1, wherein the immobilized adaptor-target-adaptors undergo a sequencing reaction from both ends to obtain a paired end read.

17. The method of claim 16, further comprising synthesizing a complementary copy of the immobilized adaptor-target-adaptors and sequencing the opposite end of the complementary copy.

18. The method of claim 17, wherein the opposite end of the complementary copy is sequenced after the sample specific tag sequence is determined.