Hi all,
I am trying to count cryptic splicing junctions using the featureCounts -J
option. The program is working great in that I am getting the same number of junctions in the output file as I see on the genome browser screen. However, a large number of my junction counts have a strand designation of "NA". I have checked individual junctions and they are not of mixed strand (i.e. all the junctions spanning a specific interval have the same orientation). The junctions I need (and the ones missing strand information) and not annotated in the GTF file. Does anyone have a fix for this problem or know what causes it?
Here is the command I am using:
featureCounts -Q 1 -J -G genome.fa --splitOnly --primary -a ~/Datafiles/human/genes.gtf -o $bam_file\_junctions $bam_file\_filtered_sorted
My libraries were made using the stranded RNAseq kit from Kapa (which give the reverse orientation to the actual transcript) and are paired-end 150bp reads. The RNA used as input was isolated nascent RNA- hence the high level of cryptic/novel splice junctions. An example from the output I am seeing is below with the problem entries in bold:
chr1 881666 881782 ENSG00000188976 33 -/
chr1 881925 883511 ENSG00000188976 20 -/
**chr1 882895 882986 ENSG00000188976 1 NA**/
chr1 883612 883870 ENSG00000188976 33 -/
chr1 883983 886507 ENSG00000188976 29 -/
chr1 886618 887380 ENSG00000188976 22 -/
chr1 887519 887792 ENSG00000188976 20 -/
chr1 887980 888555 ENSG00000188976 27 -/
chr1 888668 889162 ENSG00000188976 15 -/
chr1 889272 889384 ENSG00000188976 14 -/
chr1 889462 891303 ENSG00000188976 14 -/
chr1 889903 891303 ENSG00000188976 2 -/
chr1 891393 891475 ENSG00000188976 24 -/
chr1 891595 892274 ENSG00000188976 43 -/
chr1 892405 892479 ENSG00000188976 35 -/
chr1 892653 894309 ENSG00000188976 20 -/
chr1 896180 896673 ENSG00000187961 2 +/
chr1 896180 897009 ENSG00000187961 1 +/
**chr1 896217 896265 ENSG00000187961 1 NA**/
**chr1 896932 897009 ENSG00000187961 10 NA**/
chr1 897130 897206 ENSG00000187961 7 +/
chr1 897851 898084 ENSG00000187961 11 +/
chr1 898293 898489 ENSG00000187961 3 +/
chr1 898297 898412 ENSG00000187961 3 +/
chr1 898297 898489 ENSG00000187961 8 +/
chr1 898633 898717 ENSG00000187961 2 +/
chr1 898884 899300 ENSG00000187961 4 +/
chr1 899388 899487 ENSG00000187961 7 +/
chr1 899547 899729 ENSG00000187961 10 +/
chr1 899560 899729 ENSG00000187961 7 +/
chr1 899910 900343 ENSG00000187961 18 +/
**chr1 900940 901053 ENSG00000187961 1 NA**/
Thanks!
Please use the formatting bar (especially the
code
option) to present your post better. You can use backticks for inline code (`text` becomestext
), or select a chunk of text and use the highlighted button to format it as a code block. I've done it for you this time.Nice. I didn't know about the code option; I'll use that in the future. Thanks so much.