read counting at the exon level

[R adele]

Which  annotation file do  I input  on featurecounts to  do  read counts at the exon level ?  I used the gencode.v19.annotation.gtf , is this the correct input file? 

[Yang LIAO]

Using "gencode.v19.annotation.gtf" (I assume that it is the "Comprehensive gene annotation" from GENCODE) is fine for exon-level read counting. The count table from featureCounts gives the numbers of reads/read-pairs assigned to each exon in this annotation file.

Please be aware that some annotation files (like the one that you use) contain very many repeated exons or largely overlapping exons, but featureCounts doesn't assign reads that overlap with multiple exons by default. On the other hand, if you allow multi-assignment (by using the "-O" option), one read is assigned to all its overlapping exons, and each exon receives one count. FeatureCounts can also give fractional counts (1/N) to each of the N overlapping exons of a read if you use the "--fraction" option. You may need to read the manual and decide which strategy is the best.