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US 12,163,179 B2Granted
Methods and systems to minimize barcode exchange
Assignee10X Gemomics, Inc.
InventorsJason Bell, Josephine Lee, Corey Nemec, Francesca Meschi
Life of this patent
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Abstract
Methods and compositions to minimize barcode exchange during the preparation of barcoded next-generation sequencing libraries prepared from a single cell. The methods utilize oligonucleotides containing a 3′-terminated blocking group or sequences that prevent amplification or extension.
Claims30
A method for nucleic acid processing, comprising:
- (a) contacting, in a bulk solution, a plurality of biological particles comprising template nucleic acid molecules with a plurality of transposase complexes comprising an adapter sequence to generate, from said template nucleic acid molecules, a plurality of adapter-flanked fragments, wherein said plurality of biological particles is a plurality of cells or a plurality of cell nuclei;
- (b) partitioning said plurality of biological particles into a plurality of partitions, wherein a partition of said plurality of partitions comprises:
- (i) a biological particle of said plurality of biological particles, wherein said biological particle comprises an adapter-flanked fragment of said plurality of adapter-flanked fragments;
- (ii) a plurality of partially double-stranded nucleic acid barcode molecules each comprising:
- (I) a first strand comprising a common barcode sequence, and
- (II) a second strand comprising (A) a 5′ overhang sequence, wherein said 5′ overhang sequence comprises a noncanonical base and
- a first sequence complementary to said adapter sequence, and (B) a second sequence complementary to a sequence of said first strand;
- (c) using said adapter-flanked fragment and a first partially double-stranded, said nucleic acid barcode molecule of said plurality of partially double-stranded nucleic acid barcode molecules to generate a barcoded nucleic acid molecule comprising a sequence of said adapter-flanked fragment and said common barcode sequence;
- and
- (d) subjecting a second partially double-stranded nucleic acid barcode molecule of said plurality of partially double-stranded nucleic acid barcode molecules to nucleic acid extension conditions comprising a polymerase, wherein said noncanonical base or a derivative thereof in said second partially double-stranded nucleic acid barcode molecule inhibits full extension of said first strand of said second partially double-stranded nucleic acid barcode molecule.
7 more elements
Dependencies are inferred from the claim text.