Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA

Volden, Roger; Palmer, Theron; Byrne, Ashley; Cole, Charles N.; Schmitz, Robert J.; Green, Edward; Vollmers, Christopher

doi:10.1073/pnas.1806447115

Cited by 209 publications

(231 citation statements)

References 26 publications

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“…These 146 R2C2 reads appeared to split into 3 major and several minor isoforms. Mandalorion identified the 3 major isoforms which confirmed a previously identified splice site in the 2nd exon of CD19 that we recently observed in single B cells 9 . Because they encode distinct proteins, these isoforms may affect whether CAR T-cells 15,16 can bind leukemia cells expressing them.…”

Section: Isoform Identification and Evaluationsupporting

confidence: 86%

“…We generated R2C2 data in four technical replicates using individual ONT MinION 9.4.1 flow cells. 1D raw reads from each flow cell were processed using C3POa tools 9 we developed previously to generate a total of 10,298,086 R2C2 consensus reads. Of these reads, 122,353 could be grouped with one or more other reads based on their UMIs and were combined into 58,893 R2C2-UMI reads.…”

Section: R2c2 Data Characteristicsmentioning

confidence: 99%

“…Approximately 200ng of total RNA was used to generate full-length cDNA using a modified Smart-seq2 protocol 9 . In short, RNA was reverse transcribed using Smartscribe RT (Clontech) and ISPCR-OligodT primer ISPCR-TSO (Table S1).…”

Section: Transcriptome Sequencing Library Preparationmentioning

confidence: 99%

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Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing

Cole

Byrne

Adams

et al. 2019

Preprint

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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.

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Section: Isoform Identification and Evaluationsupporting

confidence: 86%

Section: R2c2 Data Characteristicsmentioning

confidence: 99%

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Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing

Cole

Byrne

Adams

et al. 2019

Preprint

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“…Having successfully sorted cells into cell-types, we set out to generate high quality transcriptomes for these cell-types. First, as previously established 24 , we pooled all reads associated with the cells of each cell type and then identified transcript isoforms for each cell-type using Mandalorion [19][20][21]25 . The majority (65-70%) of isoforms generated by Mandalorion for the individual cell-types were classified by SQANTI 34 as either 'full-splice-match' or 'novel-in-catalog' which represent likely full-length isoforms.…”

Section: Generating Cell-type Specific Isoform-level Transcriptomesmentioning

confidence: 99%

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

Volden

Vollmers

2020

Preprint

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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./

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“…The ability to directly sequence full-length RNAs within individual infected cells while retaining spatial and temporal information seems like science fiction. However, the speed at which nanopore and single-cell transcriptome sequencing technologies are developing seems certain to make this a reality, and soon (57). Applying these methodologies to understanding virus-host interactions remains a formidable challenge, but the increasing integration of computational biologists into experimental biology labs raises the prospects of many exciting breakthroughs for virology using NGS methodologies.…”

Section: Where We Are and Where We Are Goingmentioning

confidence: 99%

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

Depledge

Mohr

Wilson

2019

J Virol

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Transcriptome profiling has become routine in studies of many biological processes. However, the favored approaches such as short-read Illumina RNA sequencing are giving way to long-read sequencing platforms better suited to interrogating the complex transcriptomes typical of many RNA and DNA viruses. Here, we provide a guide-tailored to molecular virologists-to the ins and outs of viral transcriptome sequencing and discuss the strengths and weaknesses of the major RNA sequencing technologies as tools to analyze the abundance and diversity of the viral transcripts made during infection.

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Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA

Cited by 209 publications

References 26 publications

Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing

Complete characterization of the human immune cell transcriptome using accurate full-length cDNA sequencing

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

Going the Distance: Optimizing RNA-Seq Strategies for Transcriptomic Analysis of Complex Viral Genomes

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