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
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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.
Code: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
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Originally posted by GenoMax View PostJust want to confirm that you are using the test data (and not your own). I will take another look later this evening at this.
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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).
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