Hi guys,
I have a raw data, which is rna-seq paired end. I have downloaded genome reference from ncbi. Then, I have mapped rna seq to genome reference through tophat2. I had some results like accepted.hits.bam. After this process, I used the cufflinks for assembly. Finally, I have a transcripts.gtf file. Firstly, I do not know how to interprate these files. Secondly, After all this process, What i should do ? I mean, What options do i have ? Well, Should i use artemis or igv ? Actually i dont really know.
Thanks in advance, your respectfully,
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young_bioinformatician
2 answers
Hello young_bioinformatician,
I have a raw data, which is rna-seq paired end. I have downloaded genome reference from ncbi. Then, I have mapped rna seq to genome reference through tophat2.
You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.
Please stop using Tophat https://t.co/Es4ohxOEyx Cole and I developed the method in *2008*. It was greatly improved in TopHat2 then HISAT & HISAT2. There is no reason to use it anymore. I have been saying this for years yet it has more citations this year than last #methodsmatter
— Lior Pachter (@lpachter) December 2, 2017
I had some results like accepted.hits.bam. After this process, I used the cufflinks for assembly. Finally, I have a transcripts.gtf file. Firstly, I do not know how to interprate these files. Secondly, After all this process, What i should do ? I mean, What options do i have ? Well, Should i use artemis or igv ? Actually i dont really know.
My advice for you is to first understand RNA-seq; what is it and why are you doing it at the first place. You are currently on 'how to do it'. So go back a little bit. Find below some useful links
Now you can run cuffdiff to find out differentially expressed transcripts (if you have proper study control which is there usually in RNA seq studies). Then you can do Gene Ontology enrichment analysis to find out which functional genes are differentially expressed. Then the list goes on. Actually, you can do a lot many things which depend on your hypothesis or rationale of the study.
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Well, what do you want to know - what is the biological question?
Well, how can i make functinonal annotation through these files ? I mean, it is not a de novo assembly. Because i have a reference. Sorry, im a newbie on this field.
You just want to annotate the sequences? Or you wish to extrapolate functional information from e.g. differential expression?
If it's the former, then you need to tell us what the organism is first, but also, if you already have a reference genome - what annotation information do you need that the reference doesn't already have?
If you want to look for variants then you don't necessarily have to do any assembly at all since you have a reference.
Some general advice for a "newbie" :) - don't worry about the what or the how, so much as the why. You can't know what analysis you need to do if you don't know what you're trying to find out.
Are you a undergrad or grad student working on a specific project, or is this some kind of homework? The thing you should learn early is that the basis of every afford is a well-defined scientific question that you want to answer. Why are you analyzing these data?
This is also a cross-post from seqanswers. Please do not do that. Many users participate in both communities and might be annoyed by it, reducing your change of proper help.
Im a grad student. Actually, this is a homework which was given by my advisor. And he didnt say anything about it. He just said that you just pull reference genome and rna-seq from the database and you need to some tools, like tophat2, bowtie, and cufflinks. And Im trying to find out this workflow and why do we analyze these data. I solved some part of it but not at all. By the way, I do not know this which is a cross-platform witih seqanswer. And I'm sorry for this mistake. Thank you for warning.