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

US11732257B2 · kind B2 · utility

Assignees

• Massachusetts Institute of Technology · US
• The General Hospital Corporation · US

Inventors

• Peter van Galen · Boston, US
• Volker Hovestadt · Boston, US
• Travis Hughes · Cambridge, US
• Marc Havens Wadsworth · Cambridge, US
• Bradley E. Bernstein · Cambridge, US
• Alexander K. Shalek · Cambridge, US
• Todd Gierahn · Brookline, US
• J. Christopher Love · Somerville, US
• Ang A. Tu · Cambridge, US

Key dates

• Filing date: Oct 23, 2018
• Grant date: Aug 22, 2023
• Expiry date: Apr 7, 2040

Classification

• Technology area (CPC G): Physics
• CPC primary: G01N33/57426
• WIPO field: Measurement
• WIPO sector: Instruments

Abstract

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.