US 11,773,441 B2
High throughput multiomics sample analysis
Christina Fan, Franklin Lakes, NJ (US); and Elisabeth Marie Walczak, Franklin Lakes, NJ (US)
Assigned to Becton, Dickinson and Company, Franklin Lakes, NJ (US)
Filed by Becton, Dickinson and Company, Franklin Lakes, NJ (US)
Filed on May 1, 2019, as Appl. No. 16/400,885.
Claims priority of provisional application 62/666,483, filed on May 3, 2018.
Prior Publication US 2019/0338357 A1, Nov. 7, 2019
Int. Cl. C12Q 1/6876 (2018.01)
CPC C12Q 1/6876 (2013.01) 20 Claims
 
1. A method of sample analysis, comprising:
permeabilizing the nucleus of a cell to generate a permeabilized nucleus;
contacting double-stranded deoxyribonucleic acid (dsDNA) from the 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, wherein the dsDNA is inside the permeabilized nucleus during said contacting;
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
wherein determining the information relating the dsDNA sequence to the structure comprises determining chromatin accessibility of the dsDNA based on the sequences of the plurality of barcoded DNA fragments in the sequencing data obtained, and
wherein determining the chromatin accessibility of the dsDNA comprises:
aligning the sequences of the plurality of barcoded DNA fragments to a reference sequence of the dsDNA, and
identifying regions of the dsDNA corresponding to the ends of barcoded DNA fragments of the plurality of barcoded DNA fragments to have an accessibility above a threshold, or determining the accessibility of regions of the dsDNA corresponding the ends of ssDNA fragments of the plurality of ssDNA fragments based on the numbers of the ssDNA fragments of the plurality of ssDNA fragments in the sequencing data.