Evaluation and application of RNA-Seq by MinION

Seki, Masahide; Katsumata, Eri; Suzuki, Ayako; Sereewattanawoot, Sarun; Sakamoto, Yoshitaka; Mizushima‐Sugano, Junko; Sugano, Sumio; Kohno, Takashi; Frith, Martin C.; Tsuchihara, Katsuya; Suzuki, Yutaka

doi:10.1093/dnares/dsy038

Cited by 51 publications

(66 citation statements)

References 26 publications

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“…Figure 5c shows the quantification at gene level. As reported in other papers [8] [5] [22] the correlation at gene level is quite good (ρ =0.78). However inter-genes repeats remains a cause of mis-quantified genes.…”

Section: Quantification Of the Expression Level Of Mouse Genessupporting

confidence: 82%

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules

Sessegolo

Cruaud

Silva

et al. 2019

Preprint

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Background: Our vision of DNA transcription and splicing has changed dramatically with the introduction of short-read sequencing. These high-throughput sequencing technologies promised to unravel the complexity of any transcriptome. Generally gene expression levels are well-captured using these technologies, but there are still remaining caveats due to the limited read length and the fact that RNA molecules had to be reverse transcribed before sequencing. Oxford Nanopore Technologies has recently launched a portable sequencer which offers the possibility of sequencing long reads and most importantly RNA molecules. Results: Here we generated a full mouse transcriptome from brain and liver using the Oxford Nanopore device. As a comparison, we sequenced RNA (RNA-Seq) and cDNA (cDNA-Seq) molecules using both long and short reads technologies. In addition, we tested the TeloPrime preparation kit, dedicated to the enrichment of full-length transcripts. Conclusions: Using spike-in data, we confirmed that expression levels are efficiently captured by cDNA-Seq using short reads. More importantly, Oxford Nanopore RNA-Seq tends to be more efficient, while cDNA-Seq appears to be more biased. We further show that the cDNA library preparation of the Nanopore protocol induces read truncation for transcripts containing stretches of A's. Furthermore, bioinformatics challenges remain ahead for quantifying at the transcript level, especially when reads are not full-length. Accurate quantification of processed pseudogenes also remains difficult, and we show that current mapping protocols which map reads to the genome largely over-estimate their expression, at the expense of their parent gene.

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Section: Quantification Of the Expression Level Of Mouse Genessupporting

confidence: 82%

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules

Sessegolo

Cruaud

Silva

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…On average, 30.5% of the PromethION reads were considered full-length ("Methods"). Although the RNA samples had been stored for 5 years, they showed minimal signs of degradation as the read lengths were comparable to those observed from other nanopore cDNA-sequencing runs 26,38,43 . We observed relatively strong gene expression correlation (average Pearson correlation 0.822) between the three samples sequenced on both the minION and promethION ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 54%

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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“…Full-length transcriptome analysis using MinION was performed as previously described 49 . RNA was extracted from lung cancer cell lines using the RNeasy Mini kit (Qiagen).…”

Section: Methodsmentioning

confidence: 99%

Long read sequencing reveals a novel class of structural aberrations in cancers: identification and characterization of cancerous local amplifications

Sakamoto

Seki

et al. 2019

Preprint

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Here we report identification of a new class of local structural aberrations in lung cancers. The whole-genome sequencing of cell lines using a long read sequencer, PromethION, demonstrated that typical cancerous mutations, such as point mutations, large deletions and gene fusions can be detected also on this platform. Unexpectedly, we revealed unique structural aberrations consisting of complex combinations of local duplications, inversions and micro deletions. We further analyzed and found that these mutations also occur in vivo, even in key cancer-related genes. These mutations may elucidate the molecular etiology of patients for whom causative cancerous events and therapeutic strategies remain elusive.

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Evaluation and application of RNA-Seq by MinION

Cited by 51 publications

References 26 publications

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules

Transcriptome profiling of mouse samples using nanopore sequencing of cDNA and RNA molecules

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

Long read sequencing reveals a novel class of structural aberrations in cancers: identification and characterization of cancerous local amplifications

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