SignificanceSubtle changes in RNA transcript isoform expression can have dramatic effects on cellular behavior in both health and disease. As such, comprehensive and quantitative analysis of isoform-level transcriptomes would open an entirely new window into cellular diversity in fields ranging from developmental to cancer biology. The Rolling Circle Amplification to Concatemeric Consensus (R2C2) method we are presenting here has sufficient throughput and accuracy to make the comprehensive and quantitative analysis of RNA transcript isoforms in bulk and single-cell samples economically feasible.

Long-read sequencing holds great potential for transcriptome analysis because it offers researchers an affordable method to annotate the transcriptomes of non-model organisms. This, in turn, will greatly benefit future work on less-researched organisms like unicellular eukaryotes that cannot rely on large consortia to generate these transcriptome annotations. However, to realize this potential, several remaining molecular and computational challenges will have to be overcome. In this review, we have outlined the limitations of short-read sequencing technology and how long-read sequencing technology overcomes these limitations. We have also highlighted the unique challenges still present for long-read sequencing technology and provided some suggestions on how to overcome these challenges going forward. This article is part of a discussion meeting issue ‘Single cell ecology’.

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms.Because of this shortfall, long-read sequencing is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms.However, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption.Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here we introduce and validate a new long-read ONT based sequencing method. At the same cost, our R olling Circle Amplification to C oncatemeric C onsensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method.These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single cell samples. Significance StatementSubtle changes in RNA transcript isoform expression can have dramatic effects on cellular behaviors in both health and disease. As such, comprehensive and quantitative analysis of isoform-level transcriptomes would open an entirely new window into cellular diversity in fields ranging from developmental to cancer biology. The R2C2 method we are presenting here is the first method with sufficient throughput and accuracy to make the comprehensive and quantitative analysis of RNA transcript isoforms in bulk and single cell samples economically feasible.

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./

ABSTRACTThe human immune system relies on highly complex and diverse transcripts and the proteins they encode. These include transcripts for Human Leukocyte Antigen (HLA) class I and II receptors which are essential for self/non-self discrimination by the immune system as well as transcripts encoding B cell and T cell receptors (BCR and TCR) which recognize, bind, and help eliminate foreign antigens.HLA genes are highly diverse within the human population with each individual possessing two of thousands of different alleles in each of the 9 major HLA genes. Determining which combination of alleles an individual possesses for each HLA gene (high-resolution HLA-typing) is essential to establish donor-recipient compatibility in organ and bone-marrow transplantations. BCR and TCR genes in turn are generated by recombining a diverse set of gene segments on the DNA level in each maturing B and T cell, respectively. This process generates adaptive immune receptor repertoires (AIRR) of composed of unique transcripts expressed by each B and T cells. These repertoires carry a vast amount of health relevant information. Both short-read RNA-seq based HLA-typing1 and adaptive immune receptor repertoire sequencing2–5 currently rely heavily on our incomplete knowledge of the genetic diversity at HLA6 and BCR/TCR loci7,8.Here we used our nanopore sequencing based Rolling Circle toConcatemeric Consensus (R2C2) protocol9 to generate over 10,000,000 full-length cDNA sequences at a median accuracy of 97.9%. We used this dataset to demonstrate that deep and accurate full-length cDNA sequencing can - in addition to providing isoform-level transcriptome analysis for over 9,000 loci - be used to generate accurate sequences of HLA alleles for HLA allele typing and discovery as well as detailed AIRR data for the analysis of the adaptive immune system without requiring specific knowledge of the diversity at HLA and BCR/TCR loci.