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US 2020/0263169 A1Published application
Hybrid targeted and whole transcriptome amplification
AssigneeBecton, Dickinson and Company
InventorsEleen Shum, Christina Fan, Elisabeth Marie Walczak
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Abstract
Disclosed herein include systems, methods, compositions, and kits for the labeling of nucleic acids targets. In some embodiments, the methods comprise the use of target-specific primers and target-non-specific primers during library preparation (e.g., hybrid library preparation). In some embodiments, the methods increase the abundance of select low abundance targets during cDNA library preparation and/or sequencing library preparation.
Claims25
A method of labeling nucleic acid targets, comprising:
- (a) hybridizing a plurality of nucleic acid targets with a plurality of oligonucleotides each comprising a first universal sequence and a barcode sequence;
- (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;
- and
- (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, the second universal sequence, a complementary sequence thereof, and/or a portion thereof, and
- (e2) primers capable of hybridizing to two or more of the plurality of nucleic acid targets, thereby generating a second plurality of barcoded amplicons comprising sequences of the two or more of the plurality of nucleic acid targets, the first universal sequence, the second universal sequence, a complementary sequence thereof, and/or a portion thereof.
5 more elements
A method of labeling nucleic acid targets, comprising:
- (a) hybridizing a plurality of nucleic acid targets with a plurality of oligonucleotides;
- (b) extending the plurality of oligonucleotides in the presence of a reverse transcriptase and a template switch oligonucleotide (TSO) to generate a plurality of first strand barcoded polynucleotides comprising the TSO or a portion thereof, wherein each of the plurality of oligonucleotides and/or the template switch oligonucleotide comprises a first universal sequence and a barcode sequence;
- (c) amplifying the plurality of first strand barcoded polynucleotides using
- (c1) primers capable of hybridizing to the first universal sequence and the TSO or the portion thereof to synthesize a first plurality of second strand barcoded polynucleotides each comprising a sequence of the nucleic acid targets, the first universal sequence, the TSO, or a portion thereof, and to generate a first plurality of double-stranded barcoded polynucleotides;
- and
- (c2) primers capable of hybridizing to two or more of the plurality of nucleic acid targets to synthesize a second plurality of second strand barcoded polynucleotides each comprising a sequence of the nucleic acid targets or a portion thereof, to generate a second plurality of double-stranded barcoded polynucleotides;
- and
- (d1) amplifying the first plurality of double-stranded barcoded polynucleotides and/or the second plurality of double-stranded barcoded polynucleotides using primers capable of hybridizing to the first universal sequence and a sequence, or a subsequence, of the TSO, thereby generating a first plurality of barcoded amplicons comprising sequences of the two or more of the plurality of nucleic acid targets, and/or a portion thereof.
5 more elements
Dependencies are inferred from the claim text.