I am working with non-human RNA-seq for the first time and this is with the Rhesus Monkey. I am having very low alignment rates after featureCounts. The out put is like so:
> Load annotation file rheMac10refGene.gtf.sorted ... Features : 47357 Meta-features : 6331 Chromosomes/contigs : 46 > Process BAM file *****.bam... Single-end reads are included. Assign alignments to features... Total alignments : 84767064 Successfully assigned alignments : 8615189 (10.2%) Running time : 4.04 minutes
So the steps I've done so far:
Downloaded the entire assembly from ftp://hgdownload.soe.ucsc.edu/goldenPath/rheMac10/bigZips/*
Converted the 2bit format to .fa using twoBitToFa tool from rsync://hgdownload.soe.ucsc.edu/genome/admin/exe/linux.x86_64/
-- this is the file, which after indexing was the reference file for HISAT2
- Next, for the GTF file I downloaded the
rheMac10.refGene.txtand converted it to a GTF file using genePredToGtf tool. Which after cleaning, supplied as the gtf file for featureCounts.
Is this the way to go? My mapping rates after HISAT2 are high and comparable to the human genome ones. What am I doing wrong?