Advances in single-cell long-read sequencing technologies

Abstract

With an increase in accuracy and throughput of long-read sequencing technologies, they are rapidly being assimilated into the single-cell sequencing pipelines. For transcriptome sequencing, these techniques provide RNA isoform-level information in addition to the gene expression profiles. Long-read sequencing technologies not only help in uncovering complex patterns of cell-type specific splicing, but also offer unprecedented insights into the origin of cellular complexity and thus potentially new avenues for drug development. Additionally, single-cell long-read DNA sequencing enables high-quality assemblies, structural variant detection, haplotype phasing, resolving high-complexity regions, and characterization of epigenetic modifications. Given that significant progress has primarily occurred in single-cell RNA isoform sequencing (scRiso-seq), this review will delve into these advancements in depth and highlight the practical considerations and operational challenges, particularly pertaining to downstream analysis. We also aim to offer a concise introduction to complementary technologies for single-cell sequencing of the genome, epigenome and epitranscriptome. We conclude by identifying certain key areas of innovation that may drive these technologies further and foster more widespread application in biomedical science.

Figure 1.

Timeline of technological advancements in single-cell long-read sequencing of the genome, transcriptome and epigenome.

Figure 2.

Pictorial representation of studies from Table 1 (24 out of 32) depicting the number of cells sequenced and the average number of FL reads obtained per cell. The number of flow cells from different long-read sequencing technologies used have also been indicated.

Figure 3.

A typical long-read single-cell RNA sequencing (aka scRiso-seq) workflow. The additional step of UMI and CB correction and assignment and the available tools for the same is highlighted.