using DEseq2 for miRNA differential expression
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@farbodemami-9257
Last seen 6.8 years ago
Iran, Islamic Republic Of

Hello,

I have used mirPRo software for miRNA mining and I have found my known miRNA from my 6 fastq files (3 males and 3 females, de novo fish miRNA-seq)

Now I have a "result_precursor.csv" file that could be used For known miRNA gene differential expression analysis as the input count table. (http://sourceforge.net/p/mirpro/wiki/User%20manual/)

please help me and tell the step by step script for doing DE analysis for my 2 conditions (with 3 biological replications for each). The head of the count table is as bellow:

 

The counts for pre-miRNAs (expression value = its mature miRNA count) in all samples              
               
id E2.trimmed count E4.trimmed count E7.trimmed count E3.trimmed count E5.trimmed count E8.trimmed count  
hsa-let-7a-1 133670 116410 176291 204288 82801 42205  
hsa-let-7a-2 133036 116101 175640 203513 82338 42095  
hsa-let-7a-3 132996 116065 175571 203447 82268 42077  
hsa-let-7b 41939 36542 42194 52775 22538 12063  
hsa-let-7c 106191 122582 157398 174820 85421 32041  

 

thanks

 

               
               
               
               
               
               
               
               
deseq2 • 4.0k views
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@ryan-c-thompson-5618
Last seen 2.1 years ago
Scripps Research, La Jolla, CA

Your data looks like it is already in the right format for analysis with DESeq2. You should just be able to follow the simplest example in the DESeq2 manual to conduct a basic two-group comparison.

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I find the DESeq2 instructions confusing, in part because the example data is far more complex, and I am not sure I can even compare just two (or more) different samples, without repeats, without treatments, conditions etc. I understand that I have to build the DESeqDataSet, but I really don't understand how to get there.from a simple table like this:

  norton AMP1
vvi-miR156 206 256
vvi-miR159 100 100
vvi-miR160 0 2
vvi-miR162 98 98
vvi-miR164 97 100
vvi-miR166 200 200

Any help for clearly a complete newbie that is not very good with R would be much appreciated!!!

Susanne

 

     
     
     
     
     
     
     
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Hi Susanne,

You've likely already addressed your question by now, but DESeq2 will not work with two samples since for 2 groups (1 replicate per 'norton' and 'AMP1' group, in your example) at least 3 samples total are required, as one of the authors of DESeq2 explains in this post: https://support.bioconductor.org/p/106403/

The examples in the DESeq2 vignettes might appear complex, but they follow best practices for RNA-seq differential expression analysis, where 3 replicates per group is considered the minimum for reasonable statistical power.

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Entering edit mode

Hi Susanne,

You've likely already addressed your question by now, but DESeq2 will not work with two samples since for 2 groups (1 replicate per 'norton' and 'AMP1' group, in your example) at least 3 samples total are required, as one of the authors of DESeq2 explains in this post: https://support.bioconductor.org/p/106403/

The examples in the DESeq2 vignettes might appear complex, but they follow best practices for RNA-seq differential expression analysis, where 3 replicates per group is considered the minimum for reasonable statistical power.

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