Poster Presentation 41st Lorne Genome Conference 2020

High-throughput single cell profiling of transcriptomes and antigen receptors of B and T lymphocytes using nanopore long-read sequencing  (#240)

Manu Singh 1 , Ghamdan Al-Eryani 1 , Shaun Carswell 1 , James Ferguson 1 , Katherine Jackson 1 , Martin Smith 1 , Fabio Luciani 2 , Alex Swarbrick 1 , Chris Goodnow 1
  1. Garvan Institute of Medical Research, Sydney, NSW, Australia
  2. Immunology, Kirby Institute for Infection and Immunity, Sydney

High throughput single cell RNA sequencing is providing revolutionary insights into biological systems and disease. However droplet microfluidic platforms typically used for this application only provide short-read sequence from one end of cDNA template, for the purpose of transcript ‘counting’. This limitation restricts their application to other genomic applications including the investigation of mRNA splicing, adaptive immune responses or somatic evolution. Conversely, platforms such as SMART-Seq that deliver full length cDNA sequence are limited by higher cost per cell and lower cell throughput. To address this challenge, we have developed a method that overcomes these challenges called Receptor And Gene Expression sequencing (RAGEseq). After partitioning single cells with the 10X Chromium platform, cDNA libraries are split and undergo hybrid capture for cDNAs of interest followed by Oxford Nanopore sequencing. Libraries are analysed in parallel with Illumina sequencing to perform transcriptome profiling and accurately resolve cell barcodes and unique molecular indices. The short read data is used to demultiplex the long read data, which in turn is used to resolve the sequence of full-length transcripts via de novo assembly, error correction, and polishing. With RAGE-Seq we demonstrate that we can generate highly accurate antigen receptor sequences and link this to gene-expression profiles at the single cell level. We further use this method to phenotype clonally related lymphocytes between tissues, identify alternately-spliced BCR transcripts encoding receptors destined for secretion versus membrane localization and reveal somatic hypermutation of B cell receptors. We describe an accessible and affordable method for high throughput deep single cell profiling, applicable to a wide diversity of biological contexts.