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**jphekman** · 07-01-2014, 12:50 PM

I am pretty new to Cufflinks as well, but since no one else has jumped in yet, I will take a shot at this.

I do see gene_id values in my transcripts.gtf files:

13 Cufflinks transcript 37401708 37410554 1000 -. gene_id "EPPK1"; transcript_id "XM_532347.2"; FPKM "1.6826560109"; frac "1.000000"; conf_lo "1.436585"; conf_hi "1.928727"; cov "2.236443"; full_read_support "yes";

My command line was:

cufflinks -p 20 -g Annotation/Genes/genes.gtf -o cufflinks-out tophat-juncs/accepted_hits.bam

Did you use -g or -G? When I initially used -G I also found that my output had no gene IDs in it.

Hope this helps,
Jessica

**SrCardgage** · 11-21-2014, 07:20 AM

think of the annotation file as a guide for what to quantify

I don't think the point of supplying the annotation files is to copy the annotation data into the output file(s). The reason I use them is to stop cufflinks from attempting to build new transcripts. Instead, I want it to stick to the known genes and quantify all their transcripts. Supplying this annotation is how to tell tophat and cufflinks what are the known genes are.

Running it this way (with GTF flag and file) should also decrease the time it takes both programs to execute. However, that should not be the reason you choose to use these flags, unless you are just prototyping the programs on a new system.

**jphekman** · 11-21-2014, 09:11 AM

Interesting. I'm curious -- how would you annotate cufflinks' output?

Jessica

**SrCardgage** · 11-21-2014, 09:17 AM

programmer's overview of cufflinks annotation

Just answering off the cuff; personally, I would study the file structure and write a script to cross-reference the gene-name or genomic location (which ever is in the Cuff* files) to RefSeq database (or gene database of your choice).

If you're not proficient with scripting, there are likely tools to do this. Look into cummeRbund, briefly described in this paper on the Tuxedo pipeline:

303 See Other

http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html

If you plan on doing a lot of analysis with Cufflinks et. al., I highly suggest becoming proficient with unix and scripting (python is a good language).

**jwhite** · 11-24-2014, 08:34 AM

Originally posted by SrCardgage View Post

I don't think the point of supplying the annotation files is to copy the annotation data into the output file(s). The reason I use them is to stop cufflinks from attempting to build new transcripts. Instead, I want it to stick to the known genes and quantify all their transcripts. Supplying this annotation is how to tell tophat and cufflinks what are the known genes are.

Running it this way (with GTF flag and file) should also decrease the time it takes both programs to execute. However, that should not be the reason you choose to use these flags, unless you are just prototyping the programs on a new system.

Thank you for pointing out that cufflinks may attempt to build new transcripts. However, I was looking for annotation to be added to my cufflinks output. Its clear that this can be done; I just needed to learn how. The -g option seems to be the answer.

jwhite

**SrCardgage** · 11-24-2014, 08:44 AM

I have to disagree. From the documentation:

"-g/--GTF-guide <reference_annotation.(gtf/gff)> Tells Cufflinks to use the supplied reference annotation (GFF) to guide RABT assembly. Reference transcripts will be tiled with faux-reads to provide additional information in assembly. Output will include all reference transcripts as well as any novel genes and isoforms that are assembled. "

RABT assembly -> Reference Annotation Based Transcript assembly

Which is described in this link.

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http://cufflinks.cbcb.umd.edu/howitworks.html#hrga

I'm sorry to sound arrogant, but you really need to sit down and pore over the documentation. Most of what you need to know is in there. The cufflinks step is more like bookkeeping for later stages. Annotation is not going to happen until the very end, when running the cummeRbund package in R.

If you are proficient in scripting and databases, you can perform your own annotation by cross-referencing the unique IDs in the cuff* output. But, why do this when the cummeRbund packages has already been written.

This is a very complex program. It's unlikely a person can just breeze through doc and get things to come out correctly. Give yourself a week to just READ. It will pay off in a huge way down the road.

Best of luck!

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