In this study, the known/model splice junctions (SJs) was defined by joining splice sites at the exon-intron boundaries of all annotated introns in the recently published genome [28 (link)], generating a total of 19,150 such model/known SJs. All other SJs involving only one or none of the known splice site were considered as novel junction. SJs in the transcriptome of V. dahliae supported by cDNA reads were identified using TopHat tool [40 (link)]. Junction reads were required to have at least 8-nt mapped on each of the adjacent exons. The junctions located inside of the coordinates of annotated genes were regarded as genic SJs. All the genic SJs were classified into one of the nine types of AS events. Seven of canonical AS events were skipped exons (ES), cassette exon (CE), alternative 5′-splice sites (A5SS), alternative 3′-splice sites (A3SS), mutually exclusive exons (MXE), alternative first exons (AFE or 5′ MXE) and alternative last exons (ALE or 3′ MXE), according to the models described previously [6 (link)]. Algorithm aJAS is based on a given gene model and calculates sequence reads supporting each distinct composite SJs associated with a specific novel SJ. The novel SJs containing at least two support reads were selected for analysis. To be a qualified candidate aJAS event, the ratio of reads supporting the novel/alternative SJs to the total was at least 15%.
Retained intron or intron retention (RI or IR) is caused by reduced usage of the candidate splice sites, which cannot be predicted effectively by considering splice junctions. This class of alternative splicing event was identified according to the border reads spanning exon-intron junction and the mean of local reads depth. Four criteria have to be met to be considered as a RI event. (1) The mean base depth in the candidate intron is at least 20% of the flanking exon; (2) The sum of the intronic depth is greater than 100; (3) border reads at either the 5′ or 3′ splice site of the candidate must be present; (4) no other type of AS event could be identified.
Retained intron or intron retention (RI or IR) is caused by reduced usage of the candidate splice sites, which cannot be predicted effectively by considering splice junctions. This class of alternative splicing event was identified according to the border reads spanning exon-intron junction and the mean of local reads depth. Four criteria have to be met to be considered as a RI event. (1) The mean base depth in the candidate intron is at least 20% of the flanking exon; (2) The sum of the intronic depth is greater than 100; (3) border reads at either the 5′ or 3′ splice site of the candidate must be present; (4) no other type of AS event could be identified.
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