OFF-TARGET CAPTURE REDUCTION IN SEQUENCING TECHNIQUES

• US 20170101676A1
• Filed: 10/05/2016
• Published: 04/13/2017
• Est. Priority Date: 10/07/2015
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Teng, Li, Hsieh, Chia-Ling, Lin, Charles, Chuang, Han-Yu
• Granted Patent: US 10,577,643 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Presented herein are methods and compositions for enhancing specific enrichment of target sequences in a nucleic acid library. Off-target hybridization probes may be used to reduce binding and/or capture of off-target regions of a nucleic acid library in a targeted sequencing workflow. The off-target hybridization probes may be specific for locations known to generate off-target sequencing reads for a particular set of hybridization probes.

Claims

1. A method of reducing off-target capture in a targeted sequencing reaction, comprising:

providing a set of off-target hybridization probes that specifically bind to a plurality of off-target sequences present in a nucleic acid library generated from a sample, the nucleic acid library comprising a plurality of nucleic acid fragments;

providing a set of target-specific hybridization probes that specifically bind to a plurality of target sequences present in the nucleic acid library;

contacting the off-target hybridization probes with the nucleic acid library under conditions whereby the off-target hybridization probes hybridize to the off-target sequences;

contacting the target-specific hybridization probes with the nucleic acid library under conditions whereby the target-specific hybridization probes hybridize to the target sequences;

selecting a group of nucleic acid fragments from the nucleic acid library bound to the target-specific hybridization probes; and

sequencing the group of nucleic acid fragments bound to the target-specific hybridization probes.

2. The method of claim 1, comprising not sequencing a different group of nucleic acid fragments bound to the off-target hybridization probes.

3. The method of claim 1, comprising sequencing the different group of nucleic acid fragments after the selecting and providing sequencing data of the different group to determine a quality metric for one or both of the target-specific hybridization probes or the off-target hybridization probes.

4. The method of claim 1, wherein the nucleic acid library comprises genomic DNA fragments and the off-target hybridization probes are specific for genomic sequences that are identified as off-target sequences.

5. The method of claim 4, wherein the genomic DNA fragments are derived from human genomic DNA.

6. The method of claim 1, wherein one or both of contacting the off-target hybridization probes with the nucleic acid library or contacting the target-specific hybridization probes with the nucleic acid library are performed in the presence of global hybridization blockers.

7. The method of claim 6, wherein the global hybridization blockers comprise one or more of Cot1 DNA, tRNA, salmon sperm DNA, poly(dIdC) and blockers targeting the universal adapters of library fragments.

8. The method of claim 1, wherein the off-target hybridization probes are specific for off-target sequences identified by assessing off-target sequence data of a reference nucleic acid library contacted with the target-specific hybridization probes.

9. The method of claim 1, wherein contacting the off-target hybridization probes with the nucleic acid library and contacting the target-specific hybridization probes with the nucleic acid library are performed at the same time.

10. The method of claim 1, wherein contacting the off-target hybridization probes with the nucleic acid library is performed before contacting the target-specific hybridization probes with the nucleic acid library.

11. The method of claim 10, wherein selecting the group of nucleic acid fragments from the nucleic acid library bound to the target-specific hybridization probes comprises removing nucleic acid fragments bound to the off-target hybridization probes before contacting the target-specific hybridization probes with the nucleic acid library.

12. The method of claim 1, wherein the off-target hybridization probes are 5′

un-modified and the target-specific hybridization probes are 5′

biotinylated.

13. The method of claim 1, wherein the off-target hybridization probes and the target-specific hybridization probes are 5′

biotinylated.

14. The method of claim 1, wherein selecting the group comprises using streptavidin beads that bind to one or both of the off-target hybridization probes or the target-specific hybridization probes.

15. The method of claim 1, wherein the target-specific hybridization probes are specific for exonic sequences and the off-target hybridization probes are specific for one or both of intronic sequences or intergenic sequences.

16. The method of claim 1, wherein the target-specific hybridization probes comprise a plurality of probes specific for a first number of different target sequences, wherein the off-target hybridization probes comprise a plurality of probes specific for a second number of different off-target hybridization sequences, and wherein the first number is greater than the second number.

17. The method of claim 16, wherein the second number is determined via a user-specified setting.

18. The method of claim 16, wherein the second number is determined based on a percentage reduction of off-target sequence reads in the sequencing data relative to a control.

19. The method of claim 1, wherein the off-target hybridization probes and the target-specific hybridization probes are single-stranded nucleic acids having lengths a range of 80 to 120 bases.

20. A method of providing probes for off-target sequence capture in a targeted sequencing reaction, comprising:

receiving a request for a set of target-specific hybridization probes;

contacting the target-specific hybridization probes with a reference nucleic acid library generated from a reference sample, the nucleic acid library comprising a plurality of nucleic acid fragments, to generate a reference group of target-specific and off-target nucleic acid fragments bound to the target-specific hybridization probes;

separating the reference group of nucleic acid fragments bound to the target-specific hybridization probes from unbound nucleic acid fragments;

sequencing the reference group of nucleic acid fragments to generate reference sequencing data;

identifying off-target sequences in the reference sequencing data; and

providing a set of off-target hybridization probes based on the identified off-target sequences.

21. The method of claim 20, wherein providing the set of off-target hybridization probes based on the identified off-target sequences comprises ranking a prevalence of a plurality of off-target sequences in the sequencing data and selecting a plurality of highest prevalence off-target sequences to design the off-target hybridization probes such that the off-target hybridization probes are specific for the highest prevalence off-target sequences.

22. The method of claim 21, wherein selecting the plurality of highest prevalence off-target sequences comprises selecting a predetermined number of off-target sequences according to the ranking.

23. The method of claim 22, wherein the predetermined number is 5000 or fewer different off-target sequences.

24. The method of claim 23, wherein the target-specific hybridization probes are specific for 10,000 or more different target sequences.

25. The method of claim 21, wherein selecting the plurality of highest prevalence off-target sequences comprises selecting a subset of off-target sequences associated with at least 50% of off-target sequence reads in the reference sequencing data.

26. The method of claim 20, wherein providing the set of off-target hybridization probes based on the identified off-target sequences comprises identifying genomic locations corresponding to off-target sequences in the reference sample and selecting a plurality of off-target sequences associated with pre-determined genomic locations to design the off-target hybridization probes such that the off-target hybridization probes are specific for the pre-determined genomic locations.

27. The method of claim 26, wherein the pre-determined genomic locations are introns or intergenic locations.

28. The method of claim 20, comprising providing the set of target-specific hybridization probes.

29. The method of claim 20, comprising receiving a request to synthesize the set of target-specific hybridization probes and synthesizing the target-specific hybridization probes.

30. The method of claim 20, wherein the set of target-specific hybridization probes is a user-defined custom set of target-specific hybridization probes.

31. The method of claim 20, comprising providing an estimated reduction in sequencing cost associated with using the off-target hybridization probes with the reference sample, wherein the estimated reduction in sequencing cost is based on a reduction in off-target sequences.

32. The method of claim 31, comprising receiving a user input comprising a sequencing budget for a user sample and providing a recommendation for using a subset of off-target hybridization probes of the off-target hybridization probes with the user sample based on the budget, a cost of sequencing with the target-specific hybridization probes, and the estimated reduction in sequencing cost.

33. The kit of claim 32, wherein the off-target hybridization probes specifically bind to intronic sequences and the target-specific hybridization probes specifically bind to exonic sequences.

34. The kit of claim 32, wherein the set of off-target hybridization probes comprises a universal set that is configured to be used with samples from a particular species.

32-1. A sequencing kit for reducing off-target capture in a targeted sequencing reaction, comprising:

a set of off-target hybridization probes that specifically bind to a plurality of off-target sequences present in a nucleic acid library generated from a sample, the nucleic acid library comprising a plurality of nucleic acid fragments; and

a set of target-specific hybridization probes that specifically bind to a plurality of target sequences present in the nucleic acid library.