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8.1 years ago
jain.ashishjain1
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10
I am trying to carry out differential expression analysis using Cuffdiff on the Bowtie aligned reads. My script is running for more than 20 hours now and its seems like Cuffdiff got stuck somewhere. Does Cuffdiff have any issues in working with aligned reads other than Tophat??
Bowtie and Tophat are different aligners used for different purposes. If you try to align RNA seq data with Bowtie, you'll lose a lot of reads since Bowtie does not account for splicing while Tophat does.
If you are not working with RNA data but you want to calculate differences for a different reason, there may be better options.
I am trying to align mouse single end RNA-Seq (3'Seq Illumina) data. In their manual they have not mentioned about any specific aligners. They just talked about aligned files (sam/bam). I am trying this because my data is not aligning well with Tophat (~50%). With Bowtie, the % of reads aligned is very good (~90%). As the alignment from Bowtie is really good, I want to use this data to get the DEGs. I am also doing this by using DESeq method but still I wanted to know why it is not working with Cuffdiff.
What kind of RNA are you trying to analyse? Has the library been PolyA selected?
It's very strange that you would get a higher %alignment with Bowtie compared to Tophat. I would suggest that you post a new question with this topic and see if anyone has experienced this before.
Have you visualised the alignments to see where the Bowtie only reads are mapping?
Hi, Jain,
Probably a lot of people have a similar question. They mix different things.
https://sites.google.com/a/brown.edu/bioinformatics-in-biomed/cufflinks-and-tophat#read
Actually there was a similar question in Biostars.org a year ago or about it.
Cuffdiff Output Interpretation
And three years ago another useful answer in the very end of the post
https://biostar.usegalaxy.org/p/5727/
As @Jotan1982 said, this is a bad idea. Bowtie2 shouldn't be used for RNA Seq data because it isn't splice aware. Tophat, STAR, HISAT2, are far better choices. I'd also consider the Alignment -> HTSeq_Count -> DESeq2 route for differential gene expression.
Hi Andrew, I am trying this because my data is not aligning well with Tophat (~50%). With Bowtie, the % of reads aligned is very good (~90%). As the alignment from Bowtie is very good, I want to use this data to get the DEGs. I am also doing this by using DESeq but still I want to know why it is not working with Cuffdiff.
I suggest you check your fastqc report to see if there are any anomalous results. Also, check what library prep was done with the person that carried out the sequencing. I'd strongly suggest you try out RNA Star, Tophat has some caveat parameters that are a bit of a pain to calculate, such as mate inner distance / mate standard deviation, Star doesn't require those, and it's recommended by GATK to align RNA reads.