US 11,732,257 B2
Single cell sequencing libraries of genomic transcript regions of interest in proximity to barcodes, and genotyping of said libraries
Peter van Galen, Boston, MA (US); Volker Hovestadt, Boston, MA (US); Travis Hughes, Cambridge, MA (US); Marc H. Wadsworth, II, Cambridge, MA (US); Bradley Bernstein, Boston, MA (US); Alexander K. Shalek, Cambridge, MA (US); Todd M. Gierahn, Cambridge, MA (US); J. Christopher Love, Cambridge, MA (US); and Ang A. Tu, Cambridge, MA (US)
Assigned to Massachusetts Institute of Technology, Cambridge, MA (US); and The General Hospital Corporation, Boston, MA (US)
Appl. No. 16/758,485
Filed by Massachusetts Institute of Technology, Cambridge, MA (US); and The General Hospital Corporation, Boston, MA (US)
PCT Filed Oct. 23, 2018, PCT No. PCT/US2018/057170
§ 371(c)(1), (2) Date Apr. 23, 2020,
PCT Pub. No. WO2019/084055, PCT Pub. Date May 2, 2019.
Claims priority of provisional application 62/575,667, filed on Oct. 23, 2017.
Prior Publication US 2020/0248175 A1, Aug. 6, 2020
Int. Cl. C12P 19/34 (2006.01); C12N 15/10 (2006.01); C12Q 1/686 (2018.01); C12Q 1/6886 (2018.01); C12Q 1/6888 (2018.01)
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
OG exemplary drawing
 
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.