I recently finished mapping an RNAseq run using STAR2.3.0 and noticed the read1 and read2 counts are different, according to samtools flagstat.

The map% is ~80% but the R1 R2 counts are:



This usually does not happen for PE sequencing, which makes me a bit nervous to proceed with analysis. What are some potential reasons something like this could occur? I do not have experience with this happening and can't seem to find reasons by searching the web.

  • $\begingroup$ Presumably, the actual read counts in your two fastq source files are the same, right? I have no experience with RNAseq but the first thing that comes to mind is a problem in the sequencing step where too many reads were of low quality and were discarded by the aligner. Does that make sense? $\endgroup$
    – terdon
    Apr 17 '19 at 18:59
  • $\begingroup$ The actual read counts of the two fastq source files are the same. It makes sense and was a thought of mine. I was wondering if there is a concrete way to check this out? $\endgroup$
    – d_kennetz
    Apr 17 '19 at 19:01
  • 1
    $\begingroup$ Use FastQC to compare the overall quality of both fastq files. $\endgroup$
    – benn
    Apr 18 '19 at 12:32
  • $\begingroup$ @d_kennetz Does the actual read counts matches R1? Have you checked which of the reads are missing in the bam file? $\endgroup$
    – Kamil S Jaron
    Apr 29 '19 at 15:59

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