Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2

Abstract

Single cell transcriptome analysis elucidates facets of cell biology that have been previously out of reach. However, the high-throughput analysis of thousands of single cell transcriptomes has been limited by sample preparation and sequencing technology. High-throughput single cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5' or 3' end. These cDNA pools are currently analyzed by short read Illumina sequencing which can identify the cellular origin of a transcript and what gene it was transcribed from. However, these methods fail to retrieve isoform information. In principle, cDNA pools prepared using these approaches can be analyzed with Pacific Biosciences and Oxford Nanopore long-read sequencers to retrieve isoform information but all current implementations rely heavily on Illumina short-reads for the analysis in addition to long reads. Here, we used R2C2 to sequence and demultiplex 9 million full-length cDNA molecules generated by the 10X Chromium platform from ~3000 peripheral blood mononuclear cells (PBMCs). We used these reads to -independent from Illumina data -cluster cells into B cells, T cells, and Monocytes and generate isoform-level transcriptomes for these cell-types. We also generated isoform-level transcriptomes for all single cells and used this information to identify a wide range of isoform diversity between genes. Finally, we also designed a computational workflow to extract paired adaptive immune receptor -T cell receptor and B cell receptor (TCR and BCR)sequences unique to each T and B cell. This work represents a new, simple, and powerful approach thatusing a single sequencing method -can extract an unprecedented amount of information from thousands of single cells./

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.…”

High throughput error corrected Nanopore single cell transcriptome sequencing

“…To assure correct barcode assignment, both studies used high accuracy Illumina sequencing data of the same libraries to guide cell barcode (cellBC) assignment to long reads. Volden et al 5 recently presented a Nanopore sequencing approach with undisclosed barcode assignment accuracy, that does not require Illumina data for cellBC assignment.…”

“…Obviously, accuracy of UMI assignment is crucial. Previous long-read single-cell sequencing approaches either did not use UMIs 4 or did not correct UMI sequencing errors 3,5 . They assigned every novel UMI read sequence to a novel UMI.…”

Characterization of a COPD-Associated NPNT Functional Splicing Genetic Variant in Human Lung Tissue via Long-Read Sequencing

“…Enriched re-amplified cDNA from two independent enrichment reactions was sequenced on a MinION 9.4.1 flow cell or a MinION 10.3 flow cell using the R2C2 method [13][14][15][16] . For each run, 1ug of DNA was prepared using the LSK-109 kit according to the manufacturer's instructions with only minor modifications.…”