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Int. J. Mol. Sci. 2017, 18(6), 1110; doi:10.3390/ijms18061110

Validation of Splicing Events in Transcriptome Sequencing Data

1
Department for Anaesthesiology, University Hospital Düsseldorf, Heinrich Heine University, 40225 Düsseldorf, Germany
2
BMFZ (Biologisch-Medizinisches Forschungszentrum), Heinrich Heine University, 40225 Düsseldorf, Germany
3
Institute of Virology, University Hospital Düsseldorf, Heinrich Heine University, 40225 Düsseldorf, Germany
4
Mathematical Institute, Heinrich Heine University, 40225 Düsseldorf, Germany
*
Author to whom correspondence should be addressed.
Academic Editor: Li Lin
Received: 5 April 2017 / Revised: 26 April 2017 / Accepted: 28 April 2017 / Published: 23 May 2017
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
View Full-Text   |   Download PDF [1035 KB, uploaded 23 May 2017]   |  

Abstract

Genomic alignments of sequenced cellular messenger RNA contain gapped alignments which are interpreted as consequence of intron removal. The resulting gap-sites, genomic locations of alignment gaps, are landmarks representing potential splice-sites. As alignment algorithms report gap-sites with a considerable false discovery rate, validations are required. We describe two quality scores, gap quality score (gqs) and weighted gap information score (wgis), developed for validation of putative splicing events: While gqs solely relies on alignment data wgis additionally considers information from the genomic sequence. FASTQ files obtained from 54 human dermal fibroblast samples were aligned against the human genome (GRCh38) using TopHat and STAR aligner. Statistical properties of gap-sites validated by gqs and wgis were evaluated by their sequence similarity to known exon-intron borders. Within the 54 samples, TopHat identifies 1,000,380 and STAR reports 6,487,577 gap-sites. Due to the lack of strand information, however, the percentage of identified GT-AG gap-sites is rather low. While gap-sites from TopHat contain ≈89% GT-AG, gap-sites from STAR only contain ≈42% GT-AG dinucleotide pairs in merged data from 54 fibroblast samples. Validation with gqs yields 156,251 gap-sites from TopHat alignments and 166,294 from STAR alignments. Validation with wgis yields 770,327 gap-sites from TopHat alignments and 1,065,596 from STAR alignments. Both alignment algorithms, TopHat and STAR, report gap-sites with considerable false discovery rate, which can drastically be reduced by validation with gqs and wgis. View Full-Text
Keywords: splice sites; RNA-seq; TopHat; STAR; MaxEnt splice sites; RNA-seq; TopHat; STAR; MaxEnt
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MDPI and ACS Style

Kaisers, W.; Ptok, J.; Schwender, H.; Schaal, H. Validation of Splicing Events in Transcriptome Sequencing Data. Int. J. Mol. Sci. 2017, 18, 1110.

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