Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome

Tilgner, Hagen; Jahanbani, Fereshteh; Gupta, Ishaan; Collier, Paul; Wei, Eric; Rasmussen, Morten; Snyder, M

doi:10.1101/gr.230516.117

Cited by 68 publications

(70 citation statements)

References 62 publications

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“…By combining that approach with microdroplets, they successfully sequenced up to 10100 million RNA molecules from 1 ng without amplification. Their studies (2,3) showed that non-random combinations are widespread, much like the patterns in human speech. Its really the fundamental justification for long-read sequencing when it comes to RNA.…”

Section: Delving Into Rnamentioning

confidence: 97%

Going Long and Direct

Webb¹

2018

BioTechniques

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Biologist Chris Vollmers of the University of California, Santa Cruz relies on RNA sequencing to study the rich and unique biology of B cells, where each cell lineage recombines its genome to produce specific antibodies. As these cells modulate their response to immune challenges, they can further mutate and produce diverse transcripts, even within the same individuals. To study these cells, short-read RNA sequencing, the current gold standard, fell short.After reverse transcription, Vollmers' team produced fulllength cDNA that they then chopped into short sequences of roughly 200 bases in length. "You have this beautiful full-length isoform information that you then lose completely because of the technology in the library preparation," Vollmers said. The resulting data was cumbersome to assemble, limiting his ability to study transcriptional isoforms and the coding regions for antibodies.So in a recent study (1), his team tried a long-read strategy. They took advantage of the full-length cDNA and used Oxford Nanopore's MinION sequencer to examine whole sequences in a single read. They soon realized that there was far more going on in B cells than they could see with short-read data. "You think you have a homogenous population, but really, cells make totally different isoforms, which may do totally different things," he said.Vollmer's work is just one example of researchers fueling sequencing innovations for mining the rich biology of RNA. Scientists are also using sequencing to find and study chemical modifications to transcripts. Routine direct RNA sequencing is

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Section: Delving Into Rnamentioning

confidence: 97%

Going Long and Direct

Webb¹

2018

BioTechniques

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“…Again, ONT sequencing enabled their long-range relationship to be resolved, while short-read sequencing produced a false-positive transcript structure in which both exons were present. We used our previously published methods 33,54 to search for similar examples of coordinated alternative exon pairing in the human genome. For example, we detected a dMEP of exons in MBNL2, a gene implicated in the development of myotonic dystrophy that overlaps a targeted haplotype block associated with alcoholism (rs9556711) 55 on chromosome 13 (Supplementary Fig.…”

Section: Using Ont Sequencing To Detect Coordination Between Distant mentioning

confidence: 99%

“…We used our previously developed scripts 33,54 to look for coordination between distant pairs of exons.…”

Section: Long-range Exon Coordination Analysismentioning

confidence: 99%

Targeted, High-Resolution Rna Sequencing of Non-Coding Genomic Regions Associated With Neuropsychiatric Functions

Hardwick

Bassett

Kaczorowski

et al. 2019

Preprint

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The human brain is one of the last frontiers of biomedical research. Genome-wide association studies (GWAS) have succeeded in identifying thousands of haplotype blocks associated with a range of neuropsychiatric traits, including disorders such as schizophrenia, Alzheimer's and Parkinson's disease. However, the majority of single nucleotide polymorphisms (SNPs) that mark these haplotype blocks fall within noncoding regions of the genome, hindering their functional validation. While some of these GWAS loci may contain cis-acting regulatory DNA elements such as enhancers, we hypothesized that many are also transcribed into non-coding RNAs that are missing from publicly available transcriptome annotations. Here, we use targeted RNA capture ('RNA CaptureSeq') in combination with nanopore long-read cDNA sequencing to transcriptionally profile 1,023 haplotype blocks across the genome containing non-coding GWAS SNPs associated with neuropsychiatric traits, using post-mortem human brain tissue from three neurologically healthy donors. We find that the majority (62%) of targeted haplotype blocks, including 13% of intergenic blocks, are transcribed into novel, multi-exonic RNAs, most of which are not yet recorded in GENCODE annotations. We validated our findings with short-read RNAseq, providing orthogonal confirmation of novel splice junctions and enabling a quantitative assessment of the long-read assemblies. Many novel transcripts are supported by independent evidence of transcription including cap analysis of gene expression (CAGE) data and epigenetic marks, and some show signs of potential functional roles. We present these transcriptomes as a preliminary atlas of noncoding transcription in human brain that can be used to connect neurological phenotypes with gene expression.2

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“…However, to accommodate Illumina sequencers' short read-length, RNA or cDNA has to be fragmented during sample preparation, thereby losing long distance RNA transcript isoform information. Specialized protocols like SLR (2) or spISO-seq (3) have been used successfully to recover long-distance information but they require either specialized instrumentation or complex workflows. The SLR method assembles mostly incomplete cDNA molecules, and has limited throughput, while spISO-seq requires a 10X Genomics instrument and generates read clouds which capture long distance information, and yet cannot assemble full-length cDNA molecules.…”

Section: Introductionmentioning

confidence: 99%

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

Volden

Palmer

Byrne

et al. 2018

Preprint

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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.

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Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome

Cited by 68 publications

References 62 publications

Going Long and Direct

Going Long and Direct

Targeted, High-Resolution Rna Sequencing of Non-Coding Genomic Regions Associated With Neuropsychiatric Functions

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

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