What's the best RNA Seq Tuxedo suite tutorial/streamlined intro protocol?

[birne412]

Hi all, new to this community. I've been attempting to learn the Tuxedo suite using their protocol "Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks." (http://www.ncbi.nlm.nih.gov/pubmed/22383036) but keep dead ending with errors. Is there a better tutorial, more up to date and streamlined tutorial out there? Thanks

[GenoMax]

What errors are you getting? I had followed the protocol with newer versions of tuxedo suite a few months ago (at least to cuffdiff part) and recall that it had worked.

[birne412]

One of the GFF ID is duplicated, but even our resident lab coder couldn't find the problem. A lot of things aren't explained all that well in the protocol, hence my troubles. I'd really like to find an additional resource to aid my understanding.

cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt

[Thu Jan 22 11:26:25 2015] Beginning transcriptome assembly merge
-------------------------------------------

[Thu Jan 22 11:26:25 2015] Preparing output location ./merged_asm/
[Thu Jan 22 11:26:29 2015] Converting GTF files to SAM
[11:26:30] Loading reference annotation.
[11:26:31] Loading reference annotation.
[11:26:33] Loading reference annotation.
[11:26:34] Loading reference annotation.
[11:26:36] Loading reference annotation.
[11:26:38] Loading reference annotation.
[Thu Jan 22 11:26:40 2015] Quantitating transcripts
Warning: Your version of Cufflinks is not up-to-date. It is recommended that you upgrade to Cufflinks v2.2.1 to benefit from the most recent features and bug fixes (http://cufflinks.cbcb.umd.edu).
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g genes.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 8 ./merged_asm/tmp/mergeSam_tmp.6.EZGXyZ 
[bam_header_read] EOF marker is absent. The input is probably truncated.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File ./merged_asm/tmp/mergeSam_tmp.6.EZGXyZ doesn't appear to be a valid BAM file, trying SAM...
[11:26:40] Loading reference annotation.
[11:26:52] Inspecting reads and determining fragment length distribution.
Processed 11167 loci.                       
> Map Properties:
>	Normalized Map Mass: 68011.00
>	Raw Map Mass: 68011.00
>	Fragment Length Distribution: Truncated Gaussian (default)
>	              Default Mean: 200
>	           Default Std Dev: 80
[11:26:54] Assembling transcripts and estimating abundances.
Processed 11167 loci.                       
[Thu Jan 22 11:27:27 2015] Comparing against reference file genes.gtf
Warning: Your version of Cufflinks is not up-to-date. It is recommended that you upgrade to Cufflinks v2.2.1 to benefit from the most recent features and bug fixes (http://cufflinks.cbcb.umd.edu).
Error: duplicate GFF ID 'FBtr0079998' encountered!
	[FAILED]
Error: could not execute cuffcompare

[GenoMax]

Just want to confirm that you are using the test data (and not your own). I will take another look later this evening at this.

[birne412]

Just want to confirm that you are using the test data (and not your own). I will take another look later this evening at this.

Yes I am.I downloaded GSE32038_simulated_fastq_files.tar.gz from http://www.ncbi.nlm.nih.gov/geo/quer...i?acc=GSE32038 and alligned it against the igenome http://support.illumina.com/sequenci...e/igenome.html BDGP5

[GenoMax]

I tried cuffmerge (using cufflinks v.2.2.1). No problems in getting the command to complete.

[birne412]

I tried cuffmerge (using cufflinks v.2.2.1). No problems in getting the command to complete.

Thanks, I'll update it. Is there an easy command for updating?

[GenoMax]

Are you administering your own machine or do you need to get someone else to do it on a central server/cluster?

[birne412]

Are you administering your own machine or do you need to get someone else to do it on a central server/cluster?

It's our lab's own mini supercomputer, so I do have root access to it

[GenoMax]

You will have to download and compile it: https://github.com/cole-trapnell-lab/cufflinks Make necessary changes to your path so you start using the new version (if you are keeping the old version around).