RNA-seq data analysis (prokaryotes) featurecounts with

Question

[Pegasus]

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,

[GenoMax]

Does your GTF have overlapping meta-features, especially gene?

[Pegasus]

Hi GenoMax,

Thanks for your reply. I tried to check the overlapped genes using kentutils tools/bedtools;

• gtfToGenePred -genePredExt file.gtf file.genePred

no exons defined for group, feature gene (perhaps try -ignoreGroupsWithoutExons)

• bedtools intersect -a file.genePred -b file.genePred -wo > overlaps.txt

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,