Methods and compositions for targeted nucleic acid sequencing

• US 10,287,623 B2
• Filed: 06/06/2016
• Issued: 05/14/2019
• Est. Priority Date: 10/29/2014
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Jarosz, Mirna, Schnall-Levin, Michael, Saxonov, Serge, Hindson, Benjamin, Zheng, Xinying
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/174,928
• Publication Number:
  US 20160281138A1
• Time in Patent Office: 1,072 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

The present invention is directed to methods, compositions and systems for capturing and analyzing sequence information contained in targeted regions of a genome. Such targeted regions may include exomes, partial exomes, introns, combinations of exonic and intronic regions, genes, panels of genes, and any other subsets of a whole genome that may be of interest.

Claims

1. A method of obtaining sequence information from one or more targeted portions of a genomic sample, the method comprising:

(a) providing a plurality of fragments from the genomic sample in discrete partitions, wherein each of the fragments further comprises a barcode, and wherein at least first and second fragments within 10 kb of each other in a contiguous sequence of the genomic sample comprise a common barcode, and wherein the discrete partitions are flowable within fluid streams;

(b) applying a library of probes directed to the one or more targeted portions of the genomic sample to the plurality of fragments;

(c) conducting a first sequencing reaction to identify sequences of the plurality of fragments that hybridized to the library of probes, thereby obtaining sequence information from the one or more targeted portions of the genomic sample;

(d) conducting a second sequencing reaction to identify sequences of fragments from the discrete partitions that did not hybridize to the library of probes.

2. The method of claim 1, wherein about 80%-99% of the probes in the library of probes are designed to hybridize to informative SNPs.

3. The method of claim 1, wherein about 65%-85% of the probes in the library of probes are designed to hybridize to informative SNPs.

4. The method of claim 1, wherein at least 65% of the probes in the library of probes are designed to hybridize to informative SNPs.

5. The method of claim 1, wherein at least 75% of the probes in the library of probes are designed to hybridize to informative SNPs.

6. The method of claim 1, wherein the one or more targeted portions comprise informative SNPs, and the informative SNPs are located within both exons and introns in the targeted portions of the genomic sample.

7. The method of claim 1, wherein the majority of the probes in the library of probes are designed to hybridize to informative SNPs spaced apart by about 1 kilobase (kb) to about 15 kb.

8. The method of claim 1, wherein a plurality of probes within the library of probes are further designed to meet one or more of the following conditions in any combination:

(i) for targeted portions of the genomic samples in which there are no informative SNPs within 10-50 kb of boundaries between exons and introns, the plurality of probes is designed to hybridize at an informative SNP within an intron from those boundaries;

(ii) for targeted portions of the genomic samples in which there is a first informative SNP within an exon and that first informative SNP is located 10-50 kb from a boundary with an adjacent intron and a second informative SNP within the adjacent intron and that second informative SNP is located 10-50 kb from the boundary, the plurality of probes is designed to hybridize to a region of the genomic sample between the first and second informative SNPs;

(iii) for targeted portions of the genomic samples comprising no informative SNPs for at least 10-50 kb, the plurality of probes is designed to hybridize every 0.5, 1, 3, or 5 kb to those targeted portions of the genomic samples;

(iv) for targeted portions of the genomic samples in which there are no informative SNPs within 10-50 kb of boundaries between exons and introns, the plurality of probes are designed to hybridize to the next closest informative SNP to the exon-intron boundaries.

9. The method of claim 1, wherein the library of probes comprises probes designed to hybridize to regions of the genomic sample that flank exons at a density that provides linkage information across barcodes.

10. The method of claim 1, wherein a range of coverage represented by the library of probes is inversely proportional to the distribution of lengths of the individual nucleic acid fragment molecules of the genomic sample in the discrete partitions, such that a higher proportion of longer individual nucleic acid fragment molecules results in use of libraries of probes with smaller ranges of coverage.

11. The method of claim 1, wherein the sequence information obtained in step (c) comprises information on one or more members of the group consisting of:

gene fusions, copy number variations, insertions, and deletions.

12. The method of claim 1, wherein the library of probes is designed to hybridize to sequences at defined distances along the genome.

13. The method of claim 1, wherein the library of probes is designed to hybridize to sequences at a random mixture of distances along the genome.

14. The method of claim 1, wherein the library of probes is designed to hybridize to sequences at one or more of 20, 50, 100, 200, 250, or 500 kb along the genome.

15. The method of claim 1, wherein the library of probes is designed to hybridize to both intronic and exonic regions of the genome.

16. The method of claim 1, wherein the method further comprises step (e) determining linkage of the two or more targeted portions of the genomic sample based upon common barcode sequences.

17. The method of claim 1, wherein the discrete partitions comprise droplets of aqueous fluid within a non-aqueous continuous phase.

18. The method of claim 1, wherein the barcode is selected from a plurality of barcodes comprising at least 100,000 different sequences.