Full-length transcript characterization of <i>SF3B1</i> 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:10.1101/410183

Cited by 63 publications

(77 citation statements)

References 46 publications

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“…FLAIR is another recent pipeline designed to identify and quantify transcripts in long-read PacBio or ONT data 39 . To compare FLAIR and TALON, we ran FLAIR on the full-length, non-chimeric PacBio reads from GM12878 replicates 1 and 2 as described in the Supplementary Methods.…”

Section: Comparison Of Talon and Flair On Gm12878 Pacbio And Ont Datamentioning

confidence: 99%

A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

Wyman

Balderrama-Gutierrez

Reese

et al. 2019

Preprint

141

147

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Alternative splicing is widely acknowledged to be a crucial regulator of gene expression and is a key contributor to both normal developmental processes and disease states. While cost-effective and accurate for quantification, short-read RNA-seq lacks the ability to resolve full-length transcript isoforms despite increasingly sophisticated computational methods. Long-read sequencing platforms such as Pacific Biosciences (PacBio) and Oxford Nanopore (ONT) bypass the transcript reconstruction challenges of short reads. Here we introduce TALON, the ENCODE4 pipeline for platform-independent analysis of long-read transcriptomes. We apply TALON to the GM12878 cell line and show that while both PacBio and ONT technologies perform well at full-transcript discovery and quantification, each displayed distinct technical artifacts. We further apply TALON to mouse hippocampus and cortex transcriptomes and find that 422 genes found in these regions have more reads associated with novel isoforms than with annotated ones. We demonstrate that TALON is a capable of tracking both known and novel transcript models as well as their expression levels across datasets for both simple studies and in larger projects. These properties will enable TALON users to move beyond the limitations of short-read data to perform isoform discovery and quantification in a uniform manner on existing and future long-read platforms.

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Section: Comparison Of Talon and Flair On Gm12878 Pacbio And Ont Datamentioning

confidence: 99%

A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

Wyman

Balderrama-Gutierrez

Reese

et al. 2019

Preprint

141

147

View full text Add to dashboard Cite

show abstract

“…However, long-read sequencing approaches offered by PacBio and Oxford nanopore are continually improving sequencing throughput and quality. Recent studies using newer Nanopore flow cell chemistry and higher-throughput platforms have demonstrated data yield orders of magnitude greater than this study (Tang et al, 2018). With greater data yield and improved transcriptome coverage, there is the potential to identify more U2AF1 S34F dysregulated isoforms with greater confidence.…”

Section: Discussionmentioning

confidence: 75%

“…Total RNA was extracted from whole cell lysate using Zymo Direct-zol RNA kits. Purified RNA was prepared for long-read following previously established protocols (Picelli et al, 2013;Byrne et al, 2017;Tang et al, 2018). Total RNA was reverse transcribed using the SmartSeq2 protocol, and amplified using 15 cycles of PCR.…”

Section: Data Generation and Processingmentioning

confidence: 99%

“…Recent studies have shown the utility of long-read approaches in capturing full-length mRNA isoforms, by constructing isoforms missed by short-read assembly methods (Oikonomopoulos et al, 2016;Byrne et al, 2017;de Jong et al, 2017;Tang et al, 2018;Workman et al, 2019). Moreover, long-read approaches have already been conducted using RNA derived from primary tumor samples harboring SF3B1 mutations, demonstrating its effectiveness in capturing mutant splicing factor transcriptome alterations (Tang et al, 2018). In addition, studies have shown the extent to which long-read data can be used as a quantitative measure for gene expression (Oikonomopoulos et al, 2016;Byrne et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Nanopore sequencing reveals U2AF1 S34F-associated full-length isoforms

Cm¹,

Hrabeta-Robinson²,

Tang

et al. 2019

Preprint

Self Cite

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U2AF1 S34F is one of the most recurrent splicing factor mutations in lung adenocarcinoma (ADC) and has been shown to cause transcriptome-wide pre-mRNA splicing alterations. While U2AF1 S34F-associated splicing alterations have been described, the function of altered mRNA isoform changes remains largely unexplored. To better understand the impact U2AF1 S34F has on isoform fate and function, we conducted high-throughput long-read cDNA sequencing from isogenic human bronchial epithelial cells with and without U2AF1 S34F mutation. We found that nearly 75% (49,366) of our long-read constructed multiexon isoforms do not overlap GENCODE or short-read assembled isoforms. We found 198 transcript isoforms with significant expression and usage changes caused by U2AF1 S34F mutation, including a novel lncRNA. Isoforms from immune-related genes were largely downregulated in mutant cells, none of which were found to have splicing changes. Finally, isoforms likely targeted by nonsense-mediated decay were preferentially downregulated in U2AF1 S34F cells, suggesting that the impact of observed isoform changes may alter the translational output of affected genes. Altogether, long-read sequencing provided additional insights into transcriptome alterations and downstream functional consequences associated with U2AF1

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“…Long-read sequencing provides accurate full-length isoform structures, but suffers from a relatively high sequencing error rate that leads to spurious splice junction detection 41 . Conversely, short-read sequencing relies on the error-prone computational reconstruction of short reads into transcript models, but has highly accurate splice junction detection 32 .…”

Section: Defining a Hybrid Transcriptomementioning

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

Cited by 63 publications

References 46 publications

A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

A technology-agnostic long-read analysis pipeline for transcriptome discovery and quantification

Nanopore sequencing reveals U2AF1 S34F-associated full-length isoforms

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

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