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Question: Tophat somehow makes Cufflinks lose ribosomal RNA
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gravatar for jaeil.han
3.0 years ago by
jaeil.han • 10
United States
jaeil.han • 10 wrote:

Hello,

I am mapping yeast RNA seq data (ribozero) using Tophat followed by Cufflinks, Cuffcompare, and Cuffdiff to see differential gene expression.

After Cuffdiff, I found that ribosomal RNA reads are all missing, while they are not missing when I used Bowtie2 for mapping. Only difference was that I used Tophat instead of Bowtie2 to integrate at strand information.

I have no idea why ribosomal RNAs are missing, and I am trying to use 'multiread correction' option in Cufflinks if I can fix it. 

Can anyone has an idea?

 

Thanks in advance!
ribosomal rna tophat cufflinks • 830 views
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modified 3.0 years ago by Jennifer Hillman Jackson ♦ 25k • written 3.0 years ago by jaeil.han • 10
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gravatar for Jennifer Hillman Jackson
3.0 years ago by
Jennifer Hillman Jackson ♦ 25k
United States
Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

Review the reference annotation used with Cuffdiff. Only transcripts represented in the reference GTF will be considered by Cuffdiff. 

If the transcripts are in the Cufflinks GTF result(s), merge those together along with any external annotation GTF datasets using Cuffmerge, then use that output as the GTF input for Cuffdiff.

Best, Jen, Galaxy team
ADD COMMENT • link written 3.0 years ago by Jennifer Hillman Jackson ♦ 25k

As an aside re: Bowtie vs Tophat. If you are using a reference GTF dataset with either, and it does not contain rRNA but is used as "truth" instead of a guide, then novel rRNA transcripts (actually, any novel transcripts) can be lost at that step. Use annotation as a guide at any of these earlier steps, or consider introducing external reference annotation later in the processing, at the Cuffmerge/Cuffdiff steps and see if that helps.

 

ADD REPLY • link written 3.0 years ago by Jennifer Hillman Jackson ♦ 25k
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