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FLAME: long-read bioinformatics tool for comprehensive spliceome characterization

Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide l...

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Autores principales: Holmqvist, Isak, Bäckerholm, Alan, Tian, Yarong, Xie, Guojiang, Thorell, Kaisa, Tang, Ka-Wei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8457008/
https://www.ncbi.nlm.nih.gov/pubmed/34253685
http://dx.doi.org/10.1261/rna.078800.121
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author Holmqvist, Isak
Bäckerholm, Alan
Tian, Yarong
Xie, Guojiang
Thorell, Kaisa
Tang, Ka-Wei
author_facet Holmqvist, Isak
Bäckerholm, Alan
Tian, Yarong
Xie, Guojiang
Thorell, Kaisa
Tang, Ka-Wei
author_sort Holmqvist, Isak
collection PubMed
description Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
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spelling pubmed-84570082021-10-01 FLAME: long-read bioinformatics tool for comprehensive spliceome characterization Holmqvist, Isak Bäckerholm, Alan Tian, Yarong Xie, Guojiang Thorell, Kaisa Tang, Ka-Wei RNA Bioinformatics Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene. Cold Spring Harbor Laboratory Press 2021-10 /pmc/articles/PMC8457008/ /pubmed/34253685 http://dx.doi.org/10.1261/rna.078800.121 Text en © 2021 Holmqvist et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society https://creativecommons.org/licenses/by-nc/4.0/This article, published in RNA, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) .
spellingShingle Bioinformatics
Holmqvist, Isak
Bäckerholm, Alan
Tian, Yarong
Xie, Guojiang
Thorell, Kaisa
Tang, Ka-Wei
FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
title FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
title_full FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
title_fullStr FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
title_full_unstemmed FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
title_short FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
title_sort flame: long-read bioinformatics tool for comprehensive spliceome characterization
topic Bioinformatics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8457008/
https://www.ncbi.nlm.nih.gov/pubmed/34253685
http://dx.doi.org/10.1261/rna.078800.121
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