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.
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