Many eukaryotic genes can give rise to different alternative transcripts depending on stage of development, cell type, and physiological cues. Current transcriptome-wide sequencing technologies highlight the remarkable extent of this regulation in metazoans and allow for RNA isoforms to be profiled in increasingly small biological samples and with a growing confidence. Understanding biological functions of sample-specific transcripts is a major challenge in genomics and RNA processing fields. Here we describe simple bioinformatics workflows that facilitate this task by streamlining reference-guided annotation of novel transcripts. A key part of our protocol is the R package factR that rapidly matches custom-assembled transcripts to their likely host genes, deduces the sequence and domain structure of novel protein products, and predicts sensitivity of newly identified RNA isoforms to nonsense-mediated decay.
