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

Tang, Alison D.; Soulette, Cameron M.; Baren, Marijke J. van; Hart, Kevyn; Hrabeta-Robinson, Eva; Wu, Catherine J.; Brooks, Angela N.

doi:10.1038/s41467-020-15171-6

Cited by 313 publications

(331 citation statements)

References 90 publications

(114 reference statements)

Supporting

Mentioning

322

Contrasting

Order By: Relevance

“…Nanopore sequencing, in contrast, has no theoretical upper limit to read length and is capable of sequencing transcripts from end to end at a single molecule level (Garalde et al 2018;Jenjaroenpun et al 2018;Workman et al 2019). Nanopore-based sequencing methods have been used to annotate transcriptome structure in a variety of organisms ranging from the relatively simple Saccharomyces cerevisiae to complex human cell lines (Byrne et al 2017;Bayega et al 2018;Garalde et al 2018;Jenjaroenpun et al 2018;Tang et al 2018;Volden et al 2018;Kadobianskyi et al 2019;Sessegolo et al 2019;Workman et al 2019). In nanoporebased direct RNA sequencing (dRNA-seq), RNA reads are captured by the 3 ′ end of their poly(A) tail and sequenced in the 3 ′ to 5 ′ direction natively, thus directly measuring the RNA molecule.…”

mentioning

confidence: 99%

The full-length transcriptome of C. elegans using direct RNA sequencing

et al. 2020

View full text Add to dashboard Cite

Current transcriptome annotations have largely relied on short read lengths intrinsic to the most widely used high-throughput cDNA sequencing technologies. For example, in the annotation of the Caenorhabditis elegans transcriptome, more than half of the transcript isoforms lack full-length support and instead rely on inference from short reads that do not span the full length of the isoform. We applied nanopore-based direct RNA sequencing to characterize the developmental polyadenylated transcriptome of C. elegans. Taking advantage of long reads spanning the full length of mRNA transcripts, we provide support for 23,865 splice isoforms across 14,611 genes, without the need for computational reconstruction of gene models. Of the isoforms identified, 3452 are novel splice isoforms not present in the WormBase WS265 annotation. Furthermore, we identified 16,342 isoforms in the 3 ′ untranslated region (3 ′ UTR), 2640 of which are novel and do not fall within 10 bp of existing 3 ′-UTR data sets and annotations. Combining 3 ′ UTRs and splice isoforms, we identified 28,858 fulllength transcript isoforms. We also determined that poly(A) tail lengths of transcripts vary across development, as do the strengths of previously reported correlations between poly(A) tail length and expression level, and poly(A) tail length and 3 ′-UTR length. Finally, we have formatted this data as a publicly accessible track hub, enabling researchers to explore this data set easily in a genome browser.

show abstract

mentioning

confidence: 99%

The full-length transcriptome of C. elegans using direct RNA sequencing

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Using these simulated datasets, the Stringtie2 study showed that Stringtie2 outperformed both FLAIR [14] and Traphlor [15]. We used the same method of assessment as was used in the Stringtie2 study.…”

Section: Benchmarking Tama and Related Softwarementioning

confidence: 99%

Illuminating the dark side of the human transcriptome with long read transcript sequencing

Kuo

Cheng

Zhang

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: The human transcriptome annotation is regarded as one of the most complete of any eukaryotic species. However, limitations in sequencing technologies have biased the annotation toward multi-exonic protein coding genes. Accurate high-throughput long read transcript sequencing can now provide stronger evidence for rare transcripts and genes such as mono-exonic and non-coding genes that were previously either undetectable or impossible to differentiate from sequencing noise. Results: We developed the Transcriptome Annotation by Modular Algorithms (TAMA) software to leverage the power of long read transcript sequencing and address the issues with current data processing pipelines. TAMA achieved high sensitivity and precision for gene and transcript model predictions in both reference guided and unguided approaches in our benchmark tests using simulated Pacific Biosciences (PacBio) and Nanopore sequencing data and real PacBio datasets. By analyzing PacBio Sequel II Iso-Seq sequencing data of the Universal Human Reference RNA (UHRR) using TAMA and other commonly used pipelines, we found that the convention of using mapping identity to measure error correction performance does not reflect actual gain in accuracy of predicted transcript models. In addition, inter-read error correction can cause major changes to read mapping, resulting in potentially over 6K erroneous gene model predictions in the Iso-Seq based human genome annotation. Using TAMA’s genome assembly based error correction and gene feature evidence, we identified 2,566 putative novel non-coding genes and 1,557 putative novel protein coding gene models.Conclusions: Long read transcript sequencing data has the power to identify novel genes within the highly annotated human genome. The use of parameter tuning and extensive output information of the TAMA software package allows for in depth exploration of eukaryotic transcriptomes. We have found long read data based evidence for thousands of unannotated genes within the human genome. More development in sequencing library preparation and data processing are required for differentiating sequencing noise from real genes in long read RNA sequencing data.

show abstract

“…Reference-based methods, such as TALON (5), compares reads to existing gene and transcript models to create novel models. Reference-free methods, such as FLAIR (6), maps reads to the reference genome, clusters alignments into groups and collapses them into isoforms. Differential transcript usage (DTU) is another transcript-level analysis that is of great interest (6)(7)(8).…”

Section: Introductionmentioning

confidence: 99%

“…Reference-free methods, such as FLAIR (6), maps reads to the reference genome, clusters alignments into groups and collapses them into isoforms. Differential transcript usage (DTU) is another transcript-level analysis that is of great interest (6)(7)(8). DTU analyses examine differences in the relative proportions of expressed isoforms between two conditions.…”

Section: Introductionmentioning

confidence: 99%

The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read tools

Dong

Tian

Gouil

et al. 2020

Preprint

View full text Add to dashboard Cite

Application of Oxford Nanopore Technologies’ long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to small library sizes and high sequence error, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopore sequencing RNA-seq datasets with the goal of obtaining gene-level and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs (“sequins”) as well as a mouse neural stem cell dataset from samples with a null mutation of the epigenetic regulator Smchd1 were analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent samples. Overall, our work shows that transcriptomic analysis of long-read nanopore data using short-read software and methods that are already in wide use can yield meaningful results.

show abstract

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

Cited by 313 publications

References 90 publications

The full-length transcriptome of C. elegans using direct RNA sequencing

The full-length transcriptome of C. elegans using direct RNA sequencing

Illuminating the dark side of the human transcriptome with long read transcript sequencing

The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read tools

Contact Info

Product

Resources

About