High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes

Abstract

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.

DENDRO: genetic heterogeneity profiling and subclone detection by single-cell RNA sequencing

“…is now commonly adopted for cancer sequencing, but we do not recommend applying DENDRO to this sequencing design because of two reasons: (1) limited number of variants can be detected with tag-based methods as they only profile a small fraction of the transcript (3-prime or 5-prime end); and (2) the sequencing depth of tag-based methods are critically low (< 0.1X), resulting in unreliable variant calling. However, we do anticipate that emerging technologies, such as long-read full-transcript scRNA-seq technologies [71] and transcriptome-based deep targeted sequencing [31,32] will overcome these limitations of tag-based scRNA-seq. Given proper experimental design, we expect that these emerging technologies will be ideally suited for the joint analysis of exonic somatic mutations and gene expression.…”

Comprehensive characterization of single cell full-length isoforms in human and mouse with long-read sequencing

“…Droplet-based methods 5 such as 10x only sequence the 3' or 5' end of transcripts which largely precludes isoform identification. Long-read sequencing can overcome this limitation and generate full-length transcript information in single cells, as illustrated in several recent studies [6][7][8][9] . However, the throughput of current long-read sequencing platforms is still not comparable to short-read platforms and the per-base accuracy is also lower, which together create many issues.…”

Exploiting B Cell Receptor Analyses to Inform on HIV-1 Vaccination Strategies

“…However, the native pairing information of heavy and light chains is essential to fully describe an individual antibody, e.g., for recombinant expression. Although pairing information can be restored to some extent from bulk analyses (i.e., by bioinformatic approaches) [56], the most robust way to achieve these information is either by single cell sorting into multi-well plates [57][58][59][60] or by co-encapsulation of single cells and RNA-capture or barcode beads (e.g., with the 10× Genomics chromium system) in picoliter droplets [23,24,[61][62][63][64][65] (Figure 3, second row). Single cell sorting into multiwell plates is typically limited in throughput to tens of thousands of cells, whereas encapsulation systems allow throughputs of hundreds of thousands of cells.…”

“…To this end, unique molecular identifiers (UMI) are introduced during cDNA generation by template-switching reverse transcription. Moreover, protocols for long read parallel sequencing (e.g., SMRT and Nanopore sequencing) have been recently applied to analyze BCR sequences [63,82].…”