High-throughput analysis of single cell transcriptomes case study

Traditionally, gene expression experiments are undertaken on samples containing millions of cells. While this allows the identification of differentially expressed genes and transcripts in distinct cell populations, many subtle differences between cells in the same sample can be overlooked. Recent advances in high-throughput cell separation and transcriptomic analysis techniques has spawned the burgeoning field of single cell transcriptomics, which allows much more detailed analysis of gene expression.

The R2C2 technique delivers highly accurate, full-length transcripts.

At the University of California, Santa Cruz, Dr. Christopher Vollmers and his team are using single-cell transcriptomics to investigate gene expression in B cells. As Dr. Vollmers points out: ‘Each B cell makes a unique antibody transcript, so you really have to go at the single cell level to understand what B cells do’ ¹.

In order to obtain full-length transcripts, which is a significant challenge when using short-read sequencing technology, the team developed a novel amplification strategy, which, when combined with the long reads delivered by nanopore sequencing, provided highly accurate, full-length reads. This Rolling Circle to Concatermeric Consensus (R2C2) method allows the generation of a consensus sequence for each transcript, thereby increasing base accuracy (Figure 1).

Utilising this technique allowed transcriptome analysis of 96 individual B cells, delivering over 400,000 full length cDNA reads with a median base accuracy of 94%². Using an updated version of their Mandalorion data analysis pipeline, these reads could be used to identify high-confidence RNA transcript isoforms. A key finding of their study was that many of the B cells analysed, which were obtained from a healthy individual, express isoforms of the CD19 gene that lack the epitope targeted by CAR T-cell therapy — a discovery which may have significant implications in cancer treatment. This finding would not have been possible using short-read sequencing or without single-cell analysis.

According to the team: ‘The R2C2 method generates a larger number of accurate reads of full-length RNA transcript isoforms than any other available longread sequencing method’ ¹. They further comment that they: ‘...believe that R2C2 has the potential to replace short-read RNA-seq and its shotgun approach to transcriptome analysis entirely, especially considering the […] wide release of the high-throughput PromethION sequencer’ ².

Figure 1: Schematic of the R2C2 method. Following cDNA circularisation, rolling circle amplification creates multiple joined copies of the transcript. After sequencing, each read is split into its constituent subreads which are then aligned to generate an accurate consensus sequence. Figure courtesy of Dr. Christopher Vollmers, University of California, Santa Cruz.

This case study is taken from the human white paper.