Massachusetts Institute of Technology

The General Hospital Corporation

Ang A Tu

J Christopher Love

Todd Michael Gierahn

Alexander K Shalek

Bradley Bernstein

Travis K Hughes

Marc H Wadsworth, Ii

Peter Van Galen

Volker Hovestadt

The present invention relates to methods of detecting region(s) of interest in a gene comprising a polyA tail. The region(s) of interest can include gene(s), region(s), mutation(s), deletion(s), insertion(s), indel(s), and/or translocation(s). The region(s) can be greater than or less than 1 kilobases from the polyA tail. Methods can include forming a library of single cell transcripts comprising the region(s) in close proximity to a cell barcode and a unique molecular identifier (UMI). Methods for distinguishing cells by genotype can include amplifying the transcripts using PCR methods and detecting the cell barcode and UMI using single cell sequencing methods. Transcripts can be enriched using tagged region-specific PCR primers. Cell barcodes can be brought into close proximity to the region(s) by circularizing the transcripts. Sequencing of the transcripts can include using primer binding sites added during PCR amplification and library indexes for multiplexed sequencing.

Single cell sequencing libraries of genomic transcript regions of interest in proximity to barcodes, and genotyping of said libraries

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Cambridge, MA, United States of America

Ang A Tu