RNA-seq data analysis (prokaryotes) featurecounts with
Hi all,
I am dealing with RNA-seq data analysis (prokaryotes). In the featureCounts step, should I include the flag;
(-O) assigns read to all their overlapping meta-features, as recommended in some discussions?
Command line used;
featureCounts -p --countReadPairs -B **-O** -C -t gene -g gene_id -a referenc-genome.gtf -o count.txt treatemnt1.sorted.bam treatemnt2.sorted.bam treatemnt3.sorted.bam treatemnt4.sorted.bam treatment5.sorted.bam control1.sorted.bam control2.sorted.bam control3.sorted.bam control4.sorted.bam control5.sorted.bam
When I included it in featureCounts, it increased the % of the successfully assigned alignments.
Thank you,
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Does your GTF have overlapping meta-features, especially
gene?Hi GenoMax,
Thanks for your reply. I tried to check the overlapped genes using kentutils tools/bedtools;
no exons defined for group, feature gene (perhaps try -ignoreGroupsWithoutExons)
unassigned_transcript_1 JAFJXZ010000001.1 + 0 237 0 237 1 0,237, 0 JYU28_00005 cmpl cmpl 0,
However, I got several errors regarding genes (without exons but CDS) presented in the gtf.file. If you recommend including the -O flag, which is the recommended (–minOverlap), since I couldn't find a recommendation in the guide ;
https://bioconductor.org/packages/release/bioc/vignettes/Rsubread/inst/doc/SubreadUsersGuide.pdf
Similar exon/CDS issue was discussed in the thread (What Does Cds And Exon Mean In A Gtf File?). I checked the genePred file, and it seems I have overlapped genes. Since it's bacteria, I believe there should be overlapped reads among the genes in the same operon. Here is head -10 for the genePred file;
unassigned_transcript_1 JAFJXZ010000001.1 + 0 237 0 237 1 0, 237, 0 JYU28_00005 cmpl cmpl 0,
unassigned_transcript_2 JAFJXZ010000001.1 + 411 934 411 934 1 411, 934, 0 JYU28_00010 cmpl cmpl 0,
unassigned_transcript_3 JAFJXZ010000010.1 - 0 224 224 224 1 0, 224, 0 JYU28_03015 none none -1,
unassigned_transcript_4 JAFJXZ010000010.1 - 479 577 577 577 1 479, 577, 0 JYU28_03020 none none -1,
unassigned_transcript_5 JAFJXZ010000010.1 - 1763 4363 4363 4363 1 1763, 4363, 0 JYU28_03025 none none -1,
unassigned_transcript_6 JAFJXZ010000011.1 + 0 453 453 453 1 0, 453, 0 JYU28_03030 none none -1,
unassigned_transcript_7 JAFJXZ010000012.1 + 0 228 228 228 1 0, 228, 0 JYU28_03035 none none -1,
unassigned_transcript_8 JAFJXZ010000012.1 + 380 497 497 497 1 380, 497, 0 JYU28_03040 none none -1,
unassigned_transcript_9 JAFJXZ010000012.1 + 711 1704 711 1704 1 711, 1704, 0 JYU28_03045 cmpl cmpl 0,
unassigned_transcript_10 JAFJXZ010000012.1 + 1833 3969 1833 3969 1 1833, 3969,0 JYU28_03050 cmpl cmpl 0,