SIRVs: Spike-In RNA Variants as External Isoform Controls in RNA-Sequencing

Paul, Lukas; Kubala, Petra; Horner, Gudrun; Ante, Michael; Hollaender, Igor; Seitz, Alexander; Reda, Torsten

doi:10.1101/080747

Cited by 38 publications

(29 citation statements)

References 24 publications

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“…We simulated a set of nanopore transcriptome reads by creating a wrapper for NanoSim 49 , a tool created for simulating genomic nanopore reads ("Methods"). The SIRV RNAs are of known sequence and concentration and are of comparable complexity to human transcripts 50 . We did not benchmark FLAIR with short-read data as Mandalorion and Pinfish do not incorporate short reads into their algorithms for a splice-site correction step.…”

Section: Productivity Analysismentioning

confidence: 99%

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

et al. 2020

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While splicing changes caused by somatic mutations in SF3B1 are known, identifying fulllength isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

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Section: Productivity Analysismentioning

confidence: 99%

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

et al. 2020

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“…We benchmarked using gffcompare [30] on assemblies using both Lexogen Spike-In RNA Variants (SIRV) sequenced with nanopore 2D technology, a simulated dataset, and using our real nanopore dataset against GENCODE transcripts. SIRV transcripts are of known sequence and concentration and are of comparable complexity to human transcripts [31]. We simulated nanopore transcriptome reads by creating a wrapper for NanoSim [32], a tool used for simulating genomic nanopore reads (Methods).…”

Section: Flair Provides Improved Full-length Isoform Detection From Lmentioning

confidence: 99%

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

Tang

Soulette

Baren

et al. 2018

Preprint

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SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia (CLL) and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 have been identified with short-read RNA sequencing, a critical barrier in understanding the functional consequences of these splicing changes is that we lack the full transcript context in which these changes are occurring. Using nanopore sequencing technology, we have resequenced full-length cDNA from CLL samples with and without the hotspot SF3B1 K700E mutation, and a normal B cell. We have developed a workflow called FLAIR (Full-Length Alternative Isoform analysis of RNA), leveraging the full-length transcript sequencing data that nanopore affords. We report results from nanopore sequencing that are concordant with known SF3B1 biology from short read sequencing as well as altered intron retention events more confidently observed using long reads. Splicing analysis of nanopore reads between the SF3B1 WT and SF3B1 K700E samples identifies alternative upstream 3' splice sites associated with SF3B1 K700E . We also find downregulation of intron retention events in SF3B1 K700E relative to SF3B1 WT and no difference between CLL SF3B1 MT and B cell, suggesting an aberrant intron retention landscape in CLL samples lacking SF3B1 mutation. With full-length isoforms, we are able to better estimate the abundance of RNA transcripts that are productive and will likely be translated versus those that are unproductive. Validation from short-read data also reveals a strong branch point sequence in these downregulated intron retention events, consistent with previously reported branch points associated with mutated SF3B1. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.

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“…For this purpose, we used Spike-in RNA Variant (SIRV) standards (Lexogen), 69 synthetic isoforms with highly complex splicing patterns from seven genes ( Fig. 2d) 31 . We synthesized OCS probes from one representative SIRV isoform per gene ("SIRV7", Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The Spike-in RNA Variant Control Mixes, or SIRV Mixes (Lexogen), are 69 synthetic transcripts from seven genes which mimic the highly complex splicing patterns found in the human transcriptome 31 . We obtained PCR products of SIRV constructs corresponding to SIRV101, SIRV201, SIRV301, SIRV403, SIRV510, SIRV601, and SIRV701.…”

Section: Sirv7mentioning

confidence: 99%

ORF Capture-Seq: a versatile method for targeted identification of full-length isoforms

Sheynkman

Tuttle

Tseng

et al. 2019

Preprint

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AbstractMost human protein-coding genes are expressed as multiple isoforms. This in turn greatly expands the functional repertoire of the encoded proteome. While at least one reliable open reading frame (ORF) model has been assigned for every gene, the majority of alternative isoforms remains uncharacterized experimentally. This is primarily due to: i) vast differences of overall levels between different isoforms expressed from common genes, and ii) the difficulty of obtaining contiguous full-length ORF sequences. Here, we present ORF Capture-Seq (OCS), a flexible and cost-effective method that addresses both challenges for targeted full-length isoform sequencing applications using collections of cloned ORFs as probes. As proof-of-concept, we show that an OCS pipeline focused on genes coding for transcription factors increases isoform detection by an order of magnitude, compared to unenriched sample. In short, OCS enables rapid discovery of isoforms from custom-selected genes and will allow mapping of the full set of human isoforms at reasonable cost.

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SIRVs: Spike-In RNA Variants as External Isoform Controls in RNA-Sequencing

Cited by 38 publications

References 24 publications

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns

ORF Capture-Seq: a versatile method for targeted identification of full-length isoforms

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