Methods for indexing samples and sequencing multiple polynucleotide templates

• US 8,053,192 B2
• Filed: 02/04/2008
• Issued: 11/08/2011
• Est. Priority Date: 02/02/2007
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Gormley, Niall Anthony, Fraser, Louise, Bignell, Helen
• Primary Examiner(s): Kim; Young J
• Application Number:
  US12/012,613
• Publication Number:
  US 20090233802A1
• Time in Patent Office: 1,373 Days
• Field of Search: None
• US Class Current: 435/6.12
• 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 preparing nucleic acid sequences for sequencing on an array and identifying subsets of nucleic acid sequences on the array, wherein each subset of nucleic acid sequences is isolated from a different source, said method comprising the steps of:

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

(b) ligating a universal adaptor to each end of each target of each sample 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 nucleic acid;

(c) amplifying the adaptor-target-adaptors with two or more sample specific amplification primers to generate amplified adaptor-target-adaptors, wherein one of said amplification primers comprises a sample specific tag sequence, and wherein said sample specific tag sequence differentiates amplified adaptor-target-adaptors originating from different samples;

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

(e) immobilizing the amplified adaptor-target-adaptors on a solid surface to generate an array of immobilized adaptor-target adaptors;

(f) sequencing the immobilized adaptor-target-adaptors on the array to determine a sequence read of each immobilized adaptor-target-adaptor comprising hybridizing a first sequencing primer to the adaptor-target-adaptors, performing a first sequencing read, and removing the first sequencing primer; and

(g) 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, thereby determining both a nucleic acid sequence of said immobilized adaptor-target-adaptors and identifying the immobilized adaptor-target-adaptors as a member of a subset of nucleic acid sequences on the array.

2. The method according to claim 1 wherein the nucleic acid target fragments can undergo a sequencing reaction from both ends to obtain a paired end read.

3. The method according to claim 2 wherein both ends of the nucleic acid fragment are sequenced before the sequence of the tag is determined.

4. The method according to claim 2 wherein the read from the second end of the fragment occurs after the tag is determined.

5. The method according to claim 1 that uses two amplification primers per sample, one of which carries the tag.

6. The method according to claim 1 that uses three amplification primers per sample, one of which carries the tag.

7. The method according to claim 1, wherein the pooled adaptor-target-adaptors are amplified on the solid surface prior to sequencing.

8. The method according to claim 7, wherein said solid surface is a collection of beads.

9. The method according to claim 8, wherein said beads are isolated as single beads in an emulsion.

10. The method according to claim 1, wherein the array of immobilized adaptor-target-adaptors is amplified prior to sequencing to generate an amplified array, and wherein said amplified array is a clustered array of amplified single molecules.

11. The method according to claim 10, wherein the clustered array is formed by solid-phase nucleic acid amplification.

12. The method according to claim 11, wherein the clustered array is formed by isothermal solid-phase nucleic acid amplification.

13. The method according to claim 1, wherein said sequencing involves cycles of ligation with labeled oligonucleotides.

14. The method according to claim 1, wherein said sequencing involves cycles of addition of nucleotides.

15. The method according to claim 14, wherein said nucleotides are labelled.

16. The method according to claim 13 or 15, wherein said labels are fluorophores.

17. The method according to claim 1, wherein the sequencing steps are carried out on an array of immobilized beads.

18. A method for sequencing nucleic acid sequences on an array and identifying subsets of nucleic acid sequences on an array, wherein each subset of nucleic acid sequences is isolated from a different source, said method comprising the steps of:

(a) providing at least two samples of randomly fragmented double stranded nucleic acid targets, wherein each sample 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 said tagged adaptor comprises a region of double stranded nucleic acid and at least one region of single stranded nucleic acid and a sample specific tag that differentiates adaptor-target-adaptors originating from different samples;

(c) pooling the adaptor-target-adaptors of different samples to generate a pooled sample of adaptor-target adaptors;

(d) amplifying the adaptor-target-adaptors with a pair of universal amplification primers to generate amplified adaptor-target-adaptors;

(e) immobilising the amplified adaptor-target-adaptors on a solid surface to generate an array of immobilised adaptor-target-adaptors;

(f) sequencing the array to determine a sequence read of each immobilised adaptor-target-adaptor comprising hybridizing a first sequencing primer to the adaptor-target-adaptors, performing a first sequencing read, and removing the first sequencing primer and;

(g) sequencing the sample specific tag sequence of each immobilised adaptor-target-adaptor by hybridizing a second sequencing primer to the adaptor-target-adaptors and performing a second sequencing read, thereby determining both a nucleic acid sequence of said immobilized adaptor-target-adaptor and identifying the immobilized adaptor-target-adaptor as a member of a subset of nucleic acids on the array.

19. The method of claim 18, wherein the sample specific tag sequence is attached directly to the target fragment in each adaptor-target-adaptor with no intervening nucleotides between the sample specific tag sequence and target fragment sequences.

20. The method according to claim 18, wherein the immobilized adaptor-target-adaptors are amplified on the solid surface prior to sequencing.

21. The method according to claim 20, wherein said solid surface is a collection of beads.

22. The method according to claim 21, wherein said beads are isolated as single beads in an emulsion.

23. The method according to claim 20, wherein the amplification generates an amplified array.

24. The method according to claim 23, wherein said amplified array is a clustered array of amplified single molecules.

25. The method according to claim 24, wherein the clustered array is formed by solid-phase nucleic acid amplification.

26. The method according to claim 25, wherein the clustered array is formed by isothermal solid-phase nucleic acid amplification.

27. The method according to claim 18, wherein said sequencing involves cycles of ligation with labeled oligonucleotides.

28. The method according to claim 18, wherein said sequencing involves cycles of addition of nucleotides.

29. The method according to claim 28, wherein said nucleotides are labelled.

30. The method according to claim 27 or 29, wherein said labels are fluorophores.

31. The method according to claim 18, wherein the sequencing step is carried out on an array of immobilized beads.

32. The method of claim 18, wherein the amplification primers are complementary to the sample specific tag sequence.

33. The method of claim 18, wherein step (d) occurs prior to step (c).