Answers.Body: <p>You have a few options:</p>
  
  <ol>
  <li>Downsample the fastq files and rerun the entire analysis. You can do this with <code>seqtk sample</code>.</li>
  <li>Downsample the BAM files, which you can do with <code>samtools view -s</code>.</li>
  <li>Divide all of the counts in the counts files by some factor and round that to an integer.</li>
  </ol>
  
  <p>I personally prefer option 2, since it's quick and doesn't usually have any side-effects unless you use two-pass alignment or did the quantification with something like salmon. Option 3 is always tempting, but I have to say I've never been convinced that this consistently produces similar enough results to options 1 or 2. </p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-25T22:22:23.527Z
Answers.Text: Small question. How to decide the fraction of reads to keep? I mean 0.2 or 0.5 or something else? On what basis I should give the fraction?
Answers.UserDisplayName: stack_learner
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-26T07:00:44.343Z
Answers.Text: You’re trying to make the library sizes similar.
Answers.UserDisplayName: Devon Ryan
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-26T08:34:19.977Z
Answers.Text: yes...so any cutoff is fine?
Answers.UserDisplayName: stack_learner
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-26T08:46:58.613Z
Answers.Text: Just eye ball a fraction from that graph.
Answers.UserDisplayName: Devon Ryan
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-24T09:32:08.660Z
Answers.Text: thanks for the reply @Devon Ryan, Yes, I feel that the 3rd option is much easier. To use the 2nd option, I have to mention that the raw read counts of the transcripts were extracted from the `stringtie` step using `prepDE.py` python script [https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual] Will there be any problem?
Answers.UserDisplayName: stack_learner
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-24T09:34:25.497Z
Answers.Text: I don't use stringTie, so I can't comment with certainty.
Answers.UserDisplayName: Devon Ryan
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-24T09:49:42.173Z
Answers.Text: ok. And if I have to use the 2nd option, Is this right? `samtools view -s number GroupA_12.bam >  GroupA_12_new.bam` What I should give the fixed number for downsampling?
Answers.UserDisplayName: stack_learner
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-24T09:51:36.203Z
Answers.Text: If you need 20% then 1234.2. 1234 is just a seed for reproducibility, `.2` is the fraction to keep.
Answers.UserDisplayName: Devon Ryan
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-04-24T07:07:45.570Z
Answers.LastActivityDate: 2020-04-24T07:07:45.570Z
Answers.UserDisplayName: Devon Ryan
Body: <p>I have 40 samples and these are into two groups. I would like to perform a differential analysis between two groups. The library size of the samples is very low. But there are two samples (GroupA_12 and GroupB_2) in which the number of counts is too large compared to other samples. Please check the library sizes below:</p>
  
  <p><a href="https://i.sstatic.net/eI2m8.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/eI2m8.png" alt="enter image description here"></a></p>
  
  <p>I wanted to downsample these two cases and then would like to perform differential analysis. May I know how to downsample those two samples. </p>
  
  <p>Any help is appreciated. thanq.</p>
  
Tags: <r><rna-seq><differential-expression><gene-expression>
Title: How to downsample some of the samples in RNA-seq data?
ThreadText: # How to downsample some of the samples in RNA-seq data?
  
  (Asked by: username_0 on 2020-04-23)
  
  I have 40 samples and these are into two groups. I would like to perform a differential analysis between two groups. The library size of the samples is very low. But there are two samples (GroupA\_12 and GroupB\_2) in which the number of counts is too large compared to other samples. Please check the library sizes below:
  
  [![enter image description here](https://i.sstatic.net/eI2m8.png)](https://i.sstatic.net/eI2m8.png)
  
  I wanted to downsample these two cases and then would like to perform differential analysis. May I know how to downsample those two samples.
  
  Any help is appreciated. thanq.
  
  **Tags:** r, rna-seq, differential-expression, gene-expression
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2020-04-24. Score: 4 | Accepted answer
  
  You have a few options:
  
  1. Downsample the fastq files and rerun the entire analysis. You can do this with `seqtk sample`.
  2. Downsample the BAM files, which you can do with `samtools view -s`.
  3. Divide all of the counts in the counts files by some factor and round that to an integer.
  
  I personally prefer option 2, since it's quick and doesn't usually have any side-effects unless you use two-pass alignment or did the quantification with something like salmon. Option 3 is always tempting, but I have to say I've never been convinced that this consistently produces similar enough results to options 1 or 2.
  
  > **Comments:**
  > **username_0**: thanks for the reply @username_1, Yes, I feel that the 3rd option is much easier. To use the 2nd option, I have to mention that the raw read counts of the transcripts were extracted from the `stringtie` step using `prepDE.py` python script [https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual] Will there be any problem?  
  > **username_1**: I don't use stringTie, so I can't comment with certainty.  
  > **username_0**: ok. And if I have to use the 2nd option, Is this right? `samtools view -s number GroupA_12.bam >  GroupA_12_new.bam` What I should give the fixed number for downsampling?  
  > **username_1**: If you need 20% then 1234.2. 1234 is just a seed for reproducibility, `.2` is the fraction to keep.  
  > **username_0**: Small question. How to decide the fraction of reads to keep? I mean 0.2 or 0.5 or something else? On what basis I should give the fraction?  
  > **username_1**: You’re trying to make the library sizes similar.  
  > **username_0**: yes...so any cutoff is fine?  
  > **username_1**: Just eye ball a fraction from that graph.  
  
  ---
  
  