Methods for indexing samples and sequencing multiple polynucleotide templates

• US 8,822,150 B2
• Filed: 04/11/2012
• Issued: 09/02/2014
• Est. Priority Date: 02/02/2007
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (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:
  US13/444,136
• Publication Number:
  US 20120316072A1
• Time in Patent Office: 874 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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) ligating to each end of each nucleic acid sequence fragment in at least two samples, each sample isolated from a different source, a universal adaptor to generate adaptor-target-adaptors, wherein said universal adaptor comprises a region of double stranded nucleic acid and at least one region of single-stranded, non-complementary nucleic acid strands, and wherein at least one of the adaptors comprises a sample specific tag sequence that differentiates adaptor-target-adaptors originating from different samples;

b) pooling the adaptor-target-adaptors of the different samples;

c) immobilizing the adaptor-target-adaptors on a surface;

d) sequencing the immobilized adaptor-target-adaptors on the surface to determine a sequence read of each immobilized adaptor-target-adaptor by hybridizing a first sequencing primer to the adaptor-target-adaptors, performing a first sequencing read and removing the first sequencing primer;

e) sequencing the sample specific tag sequence of each immobilized adaptor-target-adaptor by hybridizing a second sequencing primer to the adaptor-target-adaptors and performing a second sequencing read, wherein steps d) and e) determine a nucleic acid sequence of the immobilized adaptor-target-adaptors and identifies the immobilized adaptor-target-adaptors as a member of a subset of nucleic acid sequences.

2. The method of claim 1, wherein step (d) occurs after step (e).

3. The method of claim 1, further comprising amplifying the adaptor-target-adaptors.

4. The method of claim 1, further comprising amplifying the adaptor-target-adaptors after step (c).

5. The method of claim 1, further comprising synthesizing a complementary copy of the immobilized adaptor-target-adaptors and sequencing the complementary copy of the immobilized adaptor-target-adaptors by hybridizing a third sequencing primer to the adaptor-target-adaptors, thereby obtaining a third sequencing read.

6. The method of claim 5, wherein the sequence read of step (d) is obtained from a non-immobilized end of the nucleic acid sequence fragment.

7. 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) ligating to each end of each nucleic acid sequence fragment in at least two samples, each sample isolated from a different source, a universal adaptor to generate adaptor-target-adaptors;

b) amplifying the adaptor-target-adaptors with at least two primers to generate amplified adaptor-target-adaptors, wherein said universal adaptor comprises a region of double stranded nucleic acid and at least one region of single-stranded, non-complementary nucleic acid strands, and wherein one of the amplification primers comprises a sample specific tag sequence that differentiates amplified adaptor-target-adaptors originating from different samples;

c) pooling the amplified adaptor-target-adaptors of the different samples;

d) immobilizing the amplified adaptor-target-adaptors on a surface;

e) sequencing the immobilized adaptor-target-adaptors on the surface to determine a sequence read of each immobilized adaptor-target-adaptor by hybridizing a first sequencing primer to the adaptor-target-adaptors, performing a first sequencing read and removing the first sequencing primer;

f) sequencing the sample specific tag sequence of each immobilized adaptor-target-adaptor by hybridizing a second sequencing primer to the adaptor-target-adaptors and performing a second sequencing read, wherein steps e) and f) determine a nucleic acid sequence of the immobilized adaptor-target-adaptors and identifies the immobilized adaptor-target-adaptors as a member of a subset of nucleic acid sequences.

8. The method of claim 7, wherein step (e) occurs after step (f).

9. The method of claim 7, further comprising synthesizing a complementary copy of the immobilized adaptor-target-adaptors and sequencing the complementary copy of the immobilized adaptor-target-adaptors by hybridizing a third sequencing primer to the adaptor-target-adaptors, thereby obtaining a third sequencing read.

10. The method of claim 9, wherein the sequence read of step (e) is obtained from a non-immobilized end of the nucleic acid sequence.