US 12,071,617 B2
Hybrid targeted and whole transcriptome amplification
Eleen Shum, San Jose, CA (US); Christina Fan, San Jose, CA (US); and Elisabeth Marie Walczak, San Jose, CA (US)
Assigned to Becton, Dickinson and Company, Franklin Lakes, NJ (US)
Filed by Cellular Research, Inc., San Jose, CA (US)
Filed on Feb. 12, 2020, as Appl. No. 16/788,743.
Claims priority of provisional application 62/805,956, filed on Feb. 14, 2019.
Prior Publication US 2020/0263169 A1, Aug. 20, 2020
Int. Cl. C12N 15/10 (2006.01)
CPC C12N 15/1065 (2013.01) [C12N 15/1096 (2013.01)] 13 Claims
OG exemplary drawing
 
1. A method of labeling nucleic acid targets, the method comprising:
(a) hybridizing a plurality of nucleic acid targets with a plurality of oligonucleotides each comprising a first universal sequence and a barcode sequence, wherein the plurality of nucleic acid targets comprises two or more nucleic acid targets;
(b) extending the plurality of oligonucleotides hybridized to the plurality of nucleic acid targets to generate a plurality of first strand barcoded polynucleotides;
(c) synthesizing a plurality of second strand barcoded polynucleotides using the plurality of first strand barcoded polynucleotides as templates to generate a plurality of double-stranded barcoded polynucleotides;
(d) adding the sequence of an adaptor to the plurality of double-stranded barcoded polynucleotides, wherein the adaptor comprises a second universal sequence;
(e) amplifying the plurality of double-stranded barcoded polynucleotides using:
(e1) primers capable of hybridizing to the first universal sequence and the second universal sequence, thereby generating a first plurality of barcoded amplicons comprising sequences of the nucleic acid targets, the first universal sequence and the second universal sequence, and
(e2) primers capable of hybridizing to two or more of the plurality of nucleic acid targets and a primer capable of amplifying the first universal sequence, thereby generating a second plurality of barcoded amplicons comprising sequences of the two or more of the plurality of nucleic acid targets and the first universal sequence; and
(f) performing random priming and extension using random primers and the first plurality of barcoded amplicons and the second plurality of barcoded amplicons as templates.