US 11,773,436 B2
Using random priming to obtain full-length V(D)J information for immune repertoire sequencing
Christina Chang, San Jose, CA (US); and Margaret Nakamoto, San Jose, CA (US)
Assigned to Becton, Dickinson and Company, Franklin Lakes, NJ (US)
Filed by Becton, Dickinson and Company, Franklin Lakes, NJ (US)
Filed on Nov. 6, 2020, as Appl. No. 17/91,639.
Claims priority of provisional application 62/933,285, filed on Nov. 8, 2019.
Prior Publication US 2021/0139970 A1, May 13, 2021
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6865 (2018.01); C12Q 1/6867 (2018.01); C12Q 1/6874 (2018.01)
CPC C12Q 1/6865 (2013.01) [C12Q 1/6867 (2013.01); C12Q 1/6874 (2013.01); C12Q 2521/101 (2013.01); C12Q 2521/107 (2013.01); C12Q 2565/1025 (2013.01)] 29 Claims
 
1. A method for labeling nucleic acid targets in a sample, comprising:
contacting copies of a nucleic acid target with a plurality of oligonucleotide barcodes, wherein each oligonucleotide barcode comprises a first universal sequence, a molecular label, and a target-binding region capable of hybridizing to the nucleic acid target;
generating a plurality of barcoded nucleic acid molecules each comprising the target-binding region and a complement of the target-binding region;
hybridizing the complement of the target-binding region of each barcoded nucleic acid molecule with the target-binding region of one or more of:
(i) an oligonucleotide barcode of the plurality of oligonucleotide barcodes,
(ii) the barcoded nucleic acid molecule itself, and
(iii) a different barcoded nucleic acid molecule of the plurality of barcoded nucleic acid molecules;
extending 3′-ends of the plurality of barcoded nucleic acid molecules to generate a plurality of extended barcoded nucleic acid molecules each comprising the first molecular label and a second molecular label;
amplifying the plurality of extended barcoded nucleic acid molecules using a target-specific primer capable of hybridizing to a sequence of the nucleic acid target and a primer comprising the first universal sequence, thereby generating a first plurality of barcoded amplicons comprising the sequence of the nucleic acid target, or a portion thereof;
hybridizing random primers to the first plurality of barcoded amplicons and extending the random primers to generate a plurality of extension products, wherein the random primers comprise a second universal sequence, or a complement thereof; and
amplifying the plurality of extension products using primers capable of hybridizing to the first universal sequence and the second universal sequence, or complements thereof, thereby generating a second plurality of barcoded amplicons.