CPC C12N 15/1065 (2013.01) [C12Q 1/686 (2013.01); C12Q 1/6886 (2013.01); C12Q 1/6888 (2013.01); C12Q 2600/158 (2013.01)] | 21 Claims |
1. A method of distinguishing cells by genotype comprising:
(a) constructing a library comprising a plurality of nucleic acids wherein each nucleic acid comprises a gene comprising a polyA tail, a unique molecular identifier (UMI) and a cell barcode (cell BC) flanked by sequencing adapters at the 5′ and 3′ end,
(b) amplifying each nucleic acid in the library to create a first PCR product using a tagged 5′ primer comprising a binding site for a second PCR product and a sequence complementary to a specific gene of interest and a 3′ primer complementary to the adapter sequence at the 3′ end of the nucleic acid thereby generating a first PCR product,
(c) selectively enriching the first PCR product by binding to the tag introduced by the 5′ primer or a targeted 3′ capture with a bifunctional bead or targeted capture bead,
(d) amplifying the tag-enriched first PCR product with a 5′ primer comprising the binding site for the second PCR product and a 3′ primer complementary to the adapter sequence at the 3′ end of the nucleic acid thereby generating a second PCR product,
(e) optionally amplifying the second PCR product with a 5′ primer comprising the binding site for a third PCR product and a 3′ primer complementary to the adapter sequence at the 3′ end of the nucleic acid thereby generating the third PCR product, and
(f) determining the genotype of the cell by identifying the UMI and cell BC, thereby distinguishing the cells by genotype.
|