Body: <p>I used the <a href="https://gatk.broadinstitute.org/hc/en-us/articles/360035531192-RNAseq-short-variant-discovery-SNPs-Indels-" rel="nofollow noreferrer">GATK pipeline</a> to call SNPs on males and females using RNA-Seq data. But the males have a higher read count (~43-46M reads) than the females (~40-42M reads). This causes SNP counts to be higher for males and lower for females. This is strange because I am interested in X-chromosome and hence the question &quot;How to control for the number of reads when calling SNPs using RNA-Seq?&quot;</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-12-18T23:27:17.437Z
Comments.Text: I think there is no way to do this, the ultimate solution is to get more data. If you have more reads, you naturally have more SNPs called. You can find the intersection of males and females if you want to analyze the two together.
Comments.UserDisplayName: Phoenix Mu
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-12-19T00:56:20.470Z
Comments.Text: would it be acceptable to subsample all the reads at 40M reads with the same seed and then do the GATK pipeline? I am thinking of [seqtk](https://github.com/lh3/seqtk) to subsample.
Comments.UserDisplayName: Balan
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2021-01-01T14:19:20.860Z
Comments.Text: Some random thoughts: 
  
  A) How do you define a SNP in the X chromosome? I wouldn't expect to identify polymorphic positions in males, so I assume you call SNP a site in which the male individual is homozyogus for an allele different from the reference.
  
  B) In line with what @ATpoint suggests, give a look at how many reads are aligned in males and females (especially in X chromosome).
  
  C) Which organism is this? Are there any reasons to suspect that females are more different from the reference than males?
Comments.UserDisplayName: Fabio Marroni
Tags: <rna-seq><snp><read-mapping><reads><gatk>
Title: How to control/normalize for number of reads when calling SNPs using RNA-Seq?
ThreadText: # How to control/normalize for number of reads when calling SNPs using RNA-Seq?
  
  (Asked by: username_0 on 2020-12-18)
  
  I used the [GATK pipeline](https://gatk.broadinstitute.org/hc/en-us/articles/360035531192-RNAseq-short-variant-discovery-SNPs-Indels-) to call SNPs on males and females using RNA-Seq data. But the males have a higher read count (~43-46M reads) than the females (~40-42M reads). This causes SNP counts to be higher for males and lower for females. This is strange because I am interested in X-chromosome and hence the question "How to control for the number of reads when calling SNPs using RNA-Seq?"
  
  **Tags:** rna-seq, snp, read-mapping, reads, gatk
  
  > **Comments:**
  > **username_1**: I think there is no way to do this, the ultimate solution is to get more data. If you have more reads, you naturally have more SNPs called. You can find the intersection of males and females if you want to analyze the two together.  
  > **username_0**: would it be acceptable to subsample all the reads at 40M reads with the same seed and then do the GATK pipeline? I am thinking of [seqtk](https://github.com/lh3/seqtk) to subsample.  
  > **username_2**: Some random thoughts:   A) How do you define a SNP in the X chromosome? I wouldn't expect to identify polymorphic positions in males, so I assume you call SNP a site in which the male individual is homozyogus for an allele different from the reference.  B) In line with what @ATpoint suggests, give a look at how many reads are aligned in males and females (especially in X chromosome).  C) Which organism is this? Are there any reasons to suspect that females are more different from the reference than males?  
  
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  ## 0 answers
  
  