I am writing to inquire about the low assignment ratio (19%) that I obtained using FeatureCounts in my RNA-seq analysis. I would like to confirm whether this is a normal result, and if possible, request your assistance in identifying possible reasons for this issue. To provide some context, I used HISAT2 to align paired-end stranded RNA-seq reads to the GRCh38 reference genome. The overall alignment rate by HISAT2 was 97%, with a multi-mapping ratio of 22% and a unique mapping rate of 72%. Based on this alignment result, I attempted to use FeatureCounts to obtain read counts from the BAM file generated by HISAT2. However, the successful assignment ratio was only about 19%. Thank you for your time and assistance.
samples are the key points in molecular biology and higher technologies. did you check for quality of your samples before going to library preparation. low quality (RIN) will give low target assignment.
you can also check after sequencing quality using multiQC tools but the worst is done.
fred
Your unique mapping rate doesn't sound bad depending on your sample and the strictness of your parameters. Have you tried this again when adjusting the -s argument?
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