Off-target capture reduction in sequencing techniques

• US 10,577,643 B2
• Filed: 10/05/2016
• Issued: 03/03/2020
• 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
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US15/286,374
• Publication Number:
  US 20170101676A1
• Time in Patent Office: 1,245 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 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 target-specific hybridization probes that specifically bind to a plurality of target sequences present in the nucleic acid library, wherein the target specific hybridization probes comprise a set of probes;

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, wherein the off-target hybridization probes are specific for off-target sequences identified by assessing off-target sequence data present in reference sequence data acquired from a reference nucleic acid library contacted with the target-specific hybridization probes, wherein the target specific hybridization probes comprise the set of probes;

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, wherein a subset of nucleic acid fragments comprise the off-target sequences, and wherein the method further comprises not sequencing the subset of nucleic acid fragments bound to the off-target hybridization probes.

3. The method of claim 1, comprising sequencing the subset of nucleic acid fragments after the selecting and providing sequencing data of the subset 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 universal adapters of library fragments.

8. 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.

9. 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.

10. The method of claim 9, 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.

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

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

biotinylated.

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

biotinylated.

13. 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.

14. 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.

15. 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.

16. The method of claim 15, wherein the second number is determined via a user-specified setting.

17. The method of claim 15, wherein the second number is determined based on an estimated percentage reduction of off-target sequence reads in the sequencing data relative to a control, wherein the estimated percentage reduction is determined based on a prevalence of off-target sequence reads corresponding to the off-target hybridization probes in the off-target sequence data.

18. 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.

19. The method of claim 1, wherein providing the 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.

20. The method of claim 1, wherein providing the off-target hybridization probes based on the identified off-target sequences comprises selecting a subset of the identified off-target sequences having less than a threshold similarity with the target sequences to design the off-target hybridization probes.

21. The method of claim 1, wherein the target-specific hybridization probes are provided based on a user input and wherein the off-target hybridization probes are designed after receiving the user input.

22. The method of claim 21, wherein the user input defines a custom set of the target-specific hybridization probes.

23. The method of claim 1, comprising receiving an estimated reduction in sequencing cost associated with use of the off-target hybridization probes.

24. The method of claim 1, wherein the reference nucleic acid library is contacted with the target-specific hybridization probes and not the off-target hybridization probes to acquire the reference sequence data.

25. The method of claim 1, wherein the reference nucleic acid library is generated from a reference sample and wherein the nucleic acid library is generated from a user sample.