Overlap of genomic elements
SPLICE-q is sensitive to the overlap of genomic elements. This means the tool takes into consideration when a genome shows overlapping features that can cause issues with a correct assignment of reads to specific introns or exons. For example, for introns overlapping exons of other genes. This is problematic due to the RNA-seq methodology’s limitation that makes it difficult to confidently determine without ambiguity to which genomic element, exon or intron, these reads should be attributed to.
Therefore, SPLICE-q allows the user to select different levels of restrictiveness for strand-specific filtering. They are:
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Level 1: keeps all introns in the genome regardless of overlaps with other genomic elements.
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Level 2: selects only introns whose splice junctions do not overlap any exon of a different gene.
- Level 3: selects only introns that do not overlap with any exon of the same or a different gene. This is the default option.
If you wish to select levels 1 or 2 for your analysis, you simply have to adjust the parameter
--FilterLevel or -f. For example:
$ SPLICE-q.py -b file.bam -g annotation.gtf -f 2This will run SPLICE-q on Level 2.