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The likeliness of multiple splice sites within these very short, and highly conserved, sORFs seems at least debatable.
Figure 1 shows examples of alternatively spliced exons examined in this study: intron retention (IR), cassette exon (CE), and multiple splice sites (MS).
Percentages are organized by exon type: constitutive exons (CS), cassette exons (CE), exons with multiple splice sites (MS), and exons with intron retention (IR).
Some fraction of the unaligned sequence reads likely derive from erroneously called or multiple splice sites in the set of gene models.
Specifically, we assembled alternative splice sites from internal exons with multiple splice sites, constitutive splice sites from internal constitutive exons, and skipped splice sites from cassette exons (see methods).
The features of the explicit alternative splicing prediction methods: k-mer counts, exon lengths, and sequence conservation are used to predict multiple splice sites and intron retention events along with cassette exons and constitutive exons.
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In D. simulans for example, 12% of the multiple splice site exons (MS in Table 1) and 8% of the exons with retained introns (IR in Table 1) were missing a splice site.
For example, in the case of multiple splice site exons with two overlapping exons, a "naive" program predicting only the input exon would achieve 50% sensitivity and 100% specificity.
Using the input exon plus the union of all three single isoform gene finders yields more of the correct multiple splice site exons (71% versus ExAlt's 67%) but at the cost of a large reduction in specificity (64% versus ExAlt's 94%).
We also considered cases with multiple alternative splice sites, when the number of alternative sites was more than two.
Unlike HERV and Alu, LINE elements tend to contain multiple potential splice sites (ESE) [ 4] and polyadenylation signals [ 5] in their sequences.
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