Question: RNA-seq analysis- HiSat2 and Cufflinks
gravatar for Lindsay44
20 months ago by
Lindsay440 wrote:


I am new with RNA-seq data analysis. I am using Cygwin software in order to create a Linux environment on my windows machine.

I indexed a reference genome and am running alignment using HiSat2. Then I will use Cuffdiff to run a differential expression analysis between data sets.

The output files from the alignment are in SAM format. However, I understand these files must be reformatted before being entered into Cuffdiff. How do I reformat this files for Cufflinks? Can someone please explain how this is done using Samtools (include command/script).

I am also aware of the fact that a gene annotation input is necessary to run the aforementioned processes. There is a gene annotation file in the correct format within the reference genome file I downloaded. Is this okay to use, or is it better (or even possible) to obtain one using Cufflinks?

And can someone write a brief synopsis of some of the programs I mentioned (Cufflinks/Cuffdiff and Samtools) and possibly include the command for Cuffdiff.

Also, how long do you think it will take to run these processes, considering the programs/software I am using?

Please answer as much as you can as soon as possible.

Thank you.

ADD COMMENTlink modified 20 months ago by Devon Ryan1.9k • written 20 months ago by Lindsay440
gravatar for Devon Ryan
20 months ago by
Devon Ryan1.9k
Devon Ryan1.9k wrote:

Since this isn't a Galaxy question you'd be better of asking it elsewhere (e.g., the general site). Having said that, the answer is:

samtools view -Sbo alignments.bam alignments.sam

Regarding the annotation, this depends only on whether you need to look for novel features or not. If you don't, then skip cufflinks and use cuffquant (or better yet, don't use cuff-anything and instead use featureCounts followed by edgeR or DESeq2 in R). BTW, stringTie is faster/better than cufflinks, should you need to look for novel features/isoforms/etc.

ADD COMMENTlink written 20 months ago by Devon Ryan1.9k
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