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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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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. -
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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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.Comment
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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 BDGP5Originally posted by GenoMax View PostComment
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I tried cuffmerge (using cufflinks v.2.2.1). No problems in getting the command to complete.Comment
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Thanks, I'll update it. Is there an easy command for updating?Originally posted by GenoMax View PostComment
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Are you administering your own machine or do you need to get someone else to do it on a central server/cluster?Comment
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It's our lab's own mini supercomputer, so I do have root access to itOriginally posted by GenoMax View PostComment
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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).Comment
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...06-18-2026, 07:11 AM -
Data variability is still an issue in sequencing technologies despite the advances in reproducibility and accuracy of these platforms. But the problem does not originate in the sequencing itself, but in the previous steps, before the sample reaches the sequencer.
The first step is collection, followed by preservation and sample preparation for analysis. Most scientists overlook those steps, but not being careful might just be skewing the experiment’s results.
...06-02-2026, 10:05 AM
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