Question: Differentially expressed microRNA analysis using bowtie2-cufflink-cuffdiff
0
gravatar for illinois.ks
3.9 years ago by
illinois.ks140
Korea, Republic Of
illinois.ks140 wrote:

Hello all, 

I am working on the differentially expressed microRNAs analysis.

I have used the bowtie2-cufflink-cuffdiff pipeline.. ( for the reference I downloaded from UCSC  site)

I have two questions. 

1) When running the cufflinks, I have ran two ways (with -g  xxx.gtf option, without -g option)

 I know it should only makes small difference. But, results look somehow different.  

Especially,   the fpkm value as well as the status of significance. So, I wil get the different DEGs  based on the existence of -g option.

Does it make sense?? 

 

2)  Second, even though it is the fastq file of miRNAs, from the output file "gene_exp.diff" of cuffdiff, I can see some of reads are mapping to genes instead of miRNAs.. I may guess it is true since our experimental data can have some of mRNAs even though it intends to include microRNAs.  

Among 83 significanlly differentially expressed ones,  only 27 are mapped to microRNAs.

Is it reasonal number?? Or Do I miss something? 

 

ADD COMMENTlink modified 3.9 years ago • written 3.9 years ago by illinois.ks140
1

you can check once without cufflinks

run featureCount/htseq-count providing only miRNA unique gtf

calculate counts per million,

you can also normalize with DEseq2, and see if you get reasonable DEGs 

ADD REPLYlink written 3.9 years ago by Manvendra Singh2.0k

In addition, among the gene_exp.diff results , there are some transcripts (genes) having status of significance is "NO" because one of fpkm of samples is zero so log fold change is -inf/inf..

In my result, I have  approximately 40 miRNAs having zero fpkm for Sample 2 (i.e log foldchange is -inf) and approximantely 90 miRNAS having zero fpkm for Sample 1 (i.e. log foldchange is in) .. 

Do I also have to include those miRNAs as Differentilaly expresed ones by recalculating the logfoldchange after replacing the zero to 1 or something else???  

 

 

ADD REPLYlink modified 3.9 years ago • written 3.9 years ago by illinois.ks140
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