A comparison of single cell capture methods and amplification strategies to pair alpha and beta

Abstract

Abstract Immune repertoire amplification of T cell receptors coupled to NGS provides detailed, sequence-level insight into the immune system which can be beneficial for the development of personalized therapies. However, information related to the cognate pairing of alpha and beta chains is lost once RNA extraction is performed on the bulk sample. We have previously presented low through-put (iPair) and high throughput methods for single cell pairing of the alpha and beta receptor chain. Here, we compare different methods to amplify and pair alpha and beta from single cells, as well as track CDR3 rankings from the same sample when sorted for iPair, high throughput pairing, and bulk repertoire. Template switch oligo (TSO) with 5′ cell barcoding, 3′ cell barcoding, and iRepertoire’s multiplex primers were used independently to amplify cDNA from the same cells. The cells are sorted CD14-CD4+ with 1% Jurkat cell line spiked in. Eight thousand cells from the same population were loaded onto 2 different platforms to capture single cells: a droplet-based capture and a microwell array capture. The results demonstrate that the dominant CDR3 pairs in single cell data (regardless of capture method) match the dominant CDR3 clones in bulk repertoire. All single cell methods compared accurately called the 1% Jurkat spike-in and the top paired CDR3.