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

<p>The map% is ~80% but the R1 R2 counts are:</p>

<p>R1=139283692</p>

<p>R2=137472495</p>

<p>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.</p>

CC BY-SA 4.0
2019-04-17T18:59:16.737Z
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?
terdon
CC BY-SA 4.0
2019-04-17T19:01:21.650Z
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?
d_kennetz
CC BY-SA 4.0
2019-04-18T12:32:35.643Z
Use FastQC to compare the overall quality of both fastq files.
benn
CC BY-SA 4.0
2019-04-29T15:59:05.650Z
@d_kennetz Does the actual read counts matches R1? Have you checked which of the reads are missing in the bam file?
Kamil S Jaron
<rna-seq><illumina><star>
What could cause differing counts of R1 and R2 in Paired End Sequencing (RNASEQ)
# What could cause differing counts of R1 and R2 in Paired End Sequencing (RNASEQ)

(Asked by: username_0 on 2019-04-17)

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:

R1=139283692

R2=137472495

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.

**Tags:** rna-seq, illumina, star
> **Comments:**
> **username_1**: 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?  
> **username_0**: 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?  
> **username_2**: Use FastQC to compare the overall quality of both fastq files.  
> **username_3**: @d_kennetz Does the actual read counts matches R1? Have you checked which of the reads are missing in the bam file?  

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## 0 answers

