Method of Producing a Normalised Nucleic Acid Library Using Solid State Capture Material

• US 20150252361A1
• Filed: 09/20/2013
• Published: 09/10/2015
• Est. Priority Date: 09/25/2012
• Status: Active Grant

• Current Assignee: Agriculture Victoria Services Pty. Ltd
• Original Assignee: Agriculture Victoria Services Pty. Ltd
• Inventors: Hayden, Matthew James, Kong Kaw Wa, Stephane Laurent
• Granted Patent: US 11,021,702 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

The present invention provides a method of preparing a nucleic acid library, which includes providing a one or more nucleic acid samples, and a one or more of samples of solid state capture material; contacting each nucleic acid sample with a sample of capture material to provide captured nucleic acid samples; and pooling the captured nucleic acid samples to provide the nucleic acid library. The method is particularly suitable for preparing nucleic acids for sequencing, especially next generation sequencing and related methods such as genotyping-by-sequencing.

Claims

1. A method of preparing a normalised nucleic acid library for containing a plurality of nucleic acid species in pre-defined relative concentrations, said method including:

providing

a plurality of samples each containing a biotinylated nucleic acid species, and

a solid state capture material including streptavidin;

contacting each of the plurality of nucleic acid-containing samples with a limiting amount of the solid state capture material to provide capture some but not all of the nucleic acids from each of the nucleic-acid containing samples; and

pooling the captured nucleic acid samples, and optionally eluting the captured nucleic acids from the solid state capture material to provide the normalised nucleic acid library containing the plurality of nucleic acid species in relative concentrations determined by the amount of solid state capture material contacted with the sample.

2. The method according to claim 1, wherein the plurality of nucleic acid-containing samples are subjected to a pre-capture PCR amplification step.

3. The method according to claim 2, wherein the pre-capture PCR amplification step includes introducing barcodes via tailed oligonucleotide primers.

4. The method according to claim 1, wherein the solid state capture material includes streptavidin-coated beads.

5. The method according to claim 1, wherein the nucleic acids are ligated to biotinylated adapter molecules.

6. The method according to claim 5, wherein the adapter molecules include PCR priming sites and the nucleic acids are subjected to a pre-capture PCR amplification step.

7. The method according to claim 5, wherein unligated adapter molecules and incompetent nucleic acids with partially ligated adapter molecules are removed prior to capture.

8. The method according to claim 7, wherein said wherein said unligated adapter molecules and incompetent nucleic acids with partially ligated adapter molecules are removed by exonuclease III digestion.

9. The method according to claim 1, wherein the nucleic acids are subject to PCR using specific tailed primers, one said tailed primers being biotinylated.

10. The method according to claim 1, wherein said contacting step includes contacting an excess of nucleic acid with a fixed amount of solid state capture material.

11. The method according to claim 1, wherein the captured nucleic acid samples are washed to substantially remove unbound nucleic acids from the samples or minimise their presence in the samples.

12. The method according to claim 1, wherein the pooled nucleic acids are eluted to generate the nucleic acid library.

13. The method according to claim 1, wherein the library is a multiplexed library.

14. The method according to claim 13, wherein the amount of each nucleic acid species in the library is substantially uniformly represented in the library.

15. The method according claim 13, wherein the nucleic acid species are present in the library in different, predetermined concentrations by capturing the nucleic acid samples species to different, predetermined amounts of capture material or by pooling different amounts of the captured nucleic acids.

16. The method according to claim 1, wherein said method is performed by a liquid handling robot.

18. A method of next generation sequencing or genotyping-by-sequencing, said method including

preparing a normalised nucleic acid library by the method according to claim 1,

amplifying and/or titrating the nucleic acid library and

then performing next-generation sequencing or genotyping-by-sequencing.

17. (canceled)

19. (canceled)