HIGH THROUGHPUT MULTIOMICS SAMPLE ANALYSIS

• US 20190338357A1
• Filed: 05/01/2019
• Published: 11/07/2019
• Est. Priority Date: 05/03/2018
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Fan, Christina, Walczak, Elisabeth Marie
• Granted Patent: US 11,773,441 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide

Abstract

Disclosed herein include systems, methods, compositions, and kits for sample analysis. Nucleic acid fragments comprising a capture sequence (or a complement thereof) can be generated from double-stranded genomic deoxyribonucleic acid (gDNA), barcoded to generate single-stranded DNA (ssDNA) fragments, and sequenced. Information relating to the gDNA (e.g., genome, chromatin accessibility, methylome) can be determined based on the sequences of the ssDNA fragments in the sequencing data obtained.

Claims

1. A method of sample analysis, comprising:

contacting double-stranded deoxyribonucleic acid (dsDNA) from a cell with a transposome, wherein the transposome comprises a double-strand nuclease configured to induce a double-stranded DNA break at a structure comprising dsDNA and two copies of an adaptor having a 5′

overhang comprising a capture sequence, to generate a plurality of overhang dsDNA fragments each comprising two copies of the 5′

overhangs;

barcoding the plurality of overhang DNA fragments using a plurality of barcodes to generate a plurality of barcoded DNA fragments, wherein each of the plurality of barcodes comprises a cell label sequence, a molecular label sequence, and the capture sequence, wherein at least two of the plurality of barcodes comprise different molecular label sequences, and wherein at least two of the plurality of barcodes comprise an identical cell label sequence;

detecting sequences of the plurality of barcoded DNA fragments; and

determining information relating the dsDNA sequences to the structure comprising dsDNA, based on sequences of the plurality of barcoded DNA fragments in the sequencing data.

2. The method of claim 1, further comprising:

contacting the plurality of overhang dsDNA fragments with a polymerase to generate a plurality of complementary dsDNA fragments each comprising a complementary sequence to at least a portion of the 5′

overhang; and

denaturing the plurality of complementary dsDNA fragments to generate a plurality of single-stranded DNA (ssDNA) fragments,

wherein the ssDNA fragments are barcoded, thereby barcoding the DNA fragments.

19. The method of claim 1, wherein the capture sequence comprises a poly(dT) region.

37. The method of claim 1, wherein determining the information relating the dsDNA sequences to the structure comprising dsDNA comprises determining methylome information of the dsDNA based on the sequences of the plurality of barcoded DNA fragments in the sequencing data obtained.

38. The method of claim 37, further comprising digesting nucleosomes associated with the dsDNA of the cell, or bisulfite conversion of cytosine bases of a plurality of single stranded DNAs of the plurality of overhang DNA fragments or plurality of nucleic acid fragments to generate a plurality of bisulfite converted ssDNA comprising uracil base.

41. The method of claim 37, wherein determining the methylome information comprises:

determining whether a position of the plurality of barcoded DNA fragments in the sequencing data has a thymine base and the corresponding position in a reference sequence of the dsDNA has a cytosine base to determine the corresponding position in the dsDNA has a methylcytosine base.

42. The method of claim 1, wherein the barcoding comprises:

stochastically barcoding the plurality of DNA fragments or the plurality of nucleic acid fragments using the plurality of barcodes to generate a plurality of stochastically barcoded ssDNA fragments, or barcoding the plurality of DNA fragments or plurality of nucleic acid fragments using the plurality of barcodes associated with a particle to generate the plurality of barcoded ssDNA fragments, wherein the barcodes associated with the particle comprise an identical cell label sequence and at least 100 different molecular label sequences.

60. The method of claim 1, wherein the dsDNA from the cell is selected from the group consisting of:

nuclear DNA, nucleolar DNA, genomic DNA, mitochondrial DNA, chloroplast DNA, construct DNA, viral DNA, or a combination of two or more of the listed items.

3. A method of sample analysis, comprising:

generating a plurality of nucleic acid fragments from double-stranded deoxyribonucleic acid (dsDNA) from a cell, wherein each of the plurality of nucleic acid fragments comprises a capture sequence, a complement of the capture sequence, a reverse complement of the capture sequence, or a combination thereof;

barcoding the plurality of nucleic acid fragments using a plurality of barcodes to generate a plurality of barcoded DNA fragments, wherein each of the plurality of barcodes comprises a cell label sequence, a molecular label sequence, and the capture sequence, wherein at least two of the plurality of barcodes comprise different molecular label sequences, and wherein at least two of the plurality of barcodes comprise an identical cell label sequence; and

detecting sequences of the plurality of barcoded DNA fragments.

5. The method of claim 3, wherein generating the plurality of nucleic acid fragments comprises:

contacting the dsDNA with a transposome, wherein the transposome comprises a double-strand nuclease configured to induce a double-stranded DNA break at a structure comprising dsDNA and two copies of an adaptor comprising the capture sequence, to generate a plurality of complementary dsDNA fragments each comprising a sequence complementary to the capture sequence

6. The method of claim 3, wherein generating the plurality of nucleic acid fragments comprises:

contacting the dsDNA with a transposome, wherein the transposome comprises a double-strand nuclease configured to induce a double-stranded DNA break at a structure comprising dsDNA and two copies of an adaptor having a 5′

overhang comprising a capture sequence, to generate a plurality of overhang dsDNA fragments each comprising two copies of the 5′

overhangs; and

contacting the plurality of overhang dsDNA fragments comprising the 5′

overhangs with a polymerase to generate a plurality of complementary dsDNA fragments each comprising a complementary sequence to at least a portion of the 5′

overhangs.

11. The method of claim 3, wherein

generating the plurality of nucleic acid fragments comprises fragmenting the dsDNA to generate a plurality of dsDNA fragments.

17. The method of claim 11, wherein generating the plurality of nucleic acid fragments comprises:

appending two copies of an adaptor comprising a sequence complementary to a capture sequence to the plurality of dsDNA fragments to generate a plurality of nucleic acid fragments.

18. The method of claim 17, wherein appending the two copies of the adaptor comprises ligating the two copies of the adaptor to the plurality of dsDNA fragments to generate the plurality of nucleic acid fragments comprising the adaptor.

4. (canceled)

7-10. -10. (canceled)

12-16. -16. (canceled)

20-31. -31. (canceled)

33-36. -36. (canceled)

39-40. -40. (canceled)

43-48. -48. (canceled)

50. (canceled)

53-59. -59. (canceled)

61-66. -66. (canceled)

67. A nucleic acid reagent comprising:

a capture sequence;

a barcode;

a primer binding site; and

a double-stranded DNA-binding agent.

68. The nucleic acid reagent of claim 67, wherein the nucleic acid reagent is plasma-membrane impermeable, or is configured to specifically bind to dead cells.

69-97. -97. (canceled)

98. A method of sample analysis, comprising:

contacting double-stranded deoxyribonucleic acid (dsDNA) from a cell with a transposome, wherein the transposome comprises a double-strand nuclease configured to induce a double-stranded DNA break at a structure comprising dsDNA and two copies of an adaptor having a 5′

overhang comprising a capture sequence to generate a plurality of overhang dsDNA fragments each comprising two copies of the 5′

overhangs;

contacting the plurality of overhang dsDNA fragments with a polymerase to generate a plurality of complementary dsDNA fragments each comprising a complementary sequence to at least a portion of each of the 5′

overhang;

denaturing the plurality of complementary dsDNA fragments to generate a plurality of single-stranded DNA (ssDNA) fragments;

barcoding the plurality of ssDNA fragments using a plurality of barcodes to generate a plurality of barcoded ssDNA fragments, wherein each of the plurality of barcodes comprises a cell label sequence, a molecular label sequence, and the capture sequence, wherein at least two of the plurality of barcodes comprise different molecular label sequences, and wherein if the plurality of barcodes comprise an identical cell label sequence;

obtaining sequencing data of the plurality of barcoded ssDNA fragments; and

quantifying a quantity of the dsDNA in the cell based on a quantity of unique molecular label sequences associated with the same cell label sequence.

99. The method of claim 98, further comprising:

capturing a ssDNA fragment of the plurality of ssDNA fragments on a solid support comprising an oligonucleotide comprising the capture sequence, the cell label sequence, and the molecular label sequence, wherein the capture sequence comprises a poly dT sequence that binds to a poly A tail on the ssDNA fragment;

extending the ssDNA fragment in the 5′

to 3′

direction to produce the barcoded ssDNA fragment, the barcoded ssDNA comprising the capture sequence, molecular label sequence, and cell label sequence;

extending the oligonucleotide in the 5′

to 3′

direction using a reverse transcriptase or polymerase or combination thereof to produce a complementary DNA strand complementary to the barcoded ssDNA;

denaturing the barcoded ssDNA and complementary DNA strand to produce single stranded sequences; and

amplifying the single stranded sequences.

100-104. -104. (canceled)

105. A kit for sample analysis, comprising:

a transposome comprising;

a double-strand nuclease configured to induce a double-stranded DNA break at a structure comprising dsDNA; and

two copies of an adaptor having a 5′

overhang comprising a capture sequence; and

a plurality of barcodes, each barcode can comprise a cell label sequence, a molecular label sequence, and the capture sequence, wherein at least two of the plurality of barcodes comprise different molecular label sequences, and wherein at least two of the plurality of barcodes comprise an identical cell label sequence.

106-112. -112. (canceled)