Alternative Splicing Detection Tool—a novel PERL algorithm for sensitive detection of splicing events, based on next-generation sequencing data analysis

Abstract

Next-generation sequencing (NGS) can provide researchers with high impact information regarding alternative splice variants or transcript identifications. However, the enormous amount of data acquired from NGS platforms make the analysis of alternative splicing events hard to accomplish. For this reason, we designed the "Alternative Splicing Detection Tool" (ASDT), an algorithm that is capable of identifying alternative splicing events, including novel ones from high-throughput NGS data. ASDT is available as a PERL script at http://aias.biol.uoa.gr/~mtheo and can be executed on any system with PERL installed. In addition to the detection of annotated and novel alternative splicing events from high-throughput NGS data, ASDT can also analyze the intronic regions of genes, thus enabling the detection of novel cryptic exons residing in annotated introns, extensions of previously annotated exons, or even intron retentions. Consequently, ASDT demonstrates many innovative and unique features that can efficiently contribute to alternative splicing analysis of NGS data.

Targeted Long-Read Sequencing Decodes the Transcriptional Atlas of the Founding RAS Gene Family Members

“…Visualization of the aligned nanopore-sequencing reads with Integrative Genomics Viewer (IGV) confirmed the annotated mRNA transcripts of KRAS, NRAS and HRAS genes with significant depth. However, a bioinformatics analysis also unveiled novel splice junctions between annotated exons of these genes, which were confirmed by our in-house developed generic splicing tool "ASDT" [42] and visualization of the mapped sequencing reads with IGV (Figure 1). Notably, due to the long-read technology that nanopore sequencing offers, the derived sequencing reads that bear the novel splice junctions covered the entire cDNA sequences from the first until the last exon of each RAS gene, thus representing novel RAS mRNA transcripts (Supplementary data).…”

“…Besides mapping with Minimap2, the detection of alternative splicing events in the created FASTQ file was also implemented with our in-house developed algorithm "ASDT", which was designed by members of our group as a generic splicing tool capable of identifying alternative splicing events and cryptic exons from high-throughput sequencing datasets [42]. Briefly, "ASDT" produces nucleotide k-mers for every possible splicing event that can occur between the annotated exons of a target gene.…”

Revised Exon Structure of l-DOPA Decarboxylase (DDC) Reveals Novel Splice Variants Associated with Colorectal Cancer Progression

“…The NGS data analysis with the use of our in-house-developed algorithm [ 39 ] revealed the existence of 3 DDC novel exons, which are incorporated in novel splice variants of DDC that are less frequent than the already annotated ones ( Figure S1 ). These 3 DDC novel exons and the 6 DDC novel exons that were discovered in our previous research study [ 20 ] were termed X1 to X9; X2, X7, and X9 were the 3 exons derived from the present study.…”

“…One of these is that the analysis of differential splicing among samples or cell lines is biased towards the more abundant transcripts, and it is, thus, highly sensitive to the sequencing depth [ 42 ]. In the present study, an in-house-developed algorithm for the analysis of the NGS data was implemented, which assisted in the discovery of these lower abundant novel exons and novel splice sites due to its high sensitivity [ 39 ]. Overall, 3 DDC novel exons emerged from this NGS data analysis.…”

“…The Torrent Suite™ 4.6 software (Ion Torrent™) was used for signal processing and basecalling. The generated FASTQ file containing the trimmed NGS reads was analyzed with the Alternative Splicing Detection Tool (ASDT) [ 39 ], an in-house-developed bioinformatic tool that facilitated the discovery of novel splice junctions and cryptic exons located in the intronic regions of the DDC gene.…”