Tightly packed genes and genes in operons

For annotated references containing genes located very close to each other, as commonly found in bacteria, including operon structures, we recommend that the option "Map genes to gene regions only (fast)" is chosen. In this case, reads that map over a gene boundary will be counted towards the expression value for the gene they best map to.

With the mapping option "Also map to inter-genic regions" selected, only reads mapping completely within a genes boundaries will be counted towards the expression value for that gene. If any part of a read maps outside a given gene's boundaries then it will be considered intergenic and will not be counted towards the expression value. For tightly packed genes, especially in cases where non-coding 5' regions are not included in the gene annotation, this can be too conservative.

In both cases, reads are only mapped to, and thus counted towards the expression value of, one gene.

If differential expression is the main interest, then whether the intergenic mapping option is chosen or not may make little difference to the final results. However, if there are short genes, where the read length exceeds the gene length in some cases, then some granularity may be lost. That is, reads mapping to short genes might not be counted at all when the "Also map to inter-genic regions" is chosen.

Please also refer to the section entitled "Genes in Operons", which is found in Calculating expression values from RNA-Seq.