Hi All, I am trying to analyse some human RNA-seq dataset which is in BAM format to generate FPKM values using cufflinks. The ensembl group have aligned the data using BWA 0.5.9 to generate these BAM datasets. I realise that TopHat alignments are optimal for Cufflinks, but is it possible for me to use cufflinks or some other tool to derive FPKM values from BWA aligned reads? I tried using cufflinks on one of the BAM files but got an error: Error running cufflinks. return code = -11 Command line: cufflinks -q --no-update-check -I 300000 -F 0.100000 -j 0.150000 -p 8 /galaxy-repl/main/files/006/853/dataset_6853120.dat The additional output was: cufflinks v2.1.1 cufflinks -q --no-update-check -I 300000 -F 0.100000 -j 0.150000 -p 8 I have sent it to the galaxy team and awaiting their reply. Any thoughts about how I can go about analysing this data. Best wishes Mervyn Dr. Mervyn G Thomas BSc, MBChB, PhD Academic Foundation Doctor School of Medicine University of Leicester UK
Hello Mervyn, I just replied to your submission to the bugs list. For all in general - alternate mapping tools can be used but which are appropriate depend on the inputs. If the transcriptome is unspliced (bacterial, etc), then Bowtie is often used. Just be sure to sort the resulting file before submitting to Cufflinks. If the transcriptome is spliced, then the mapper should map spliced reads, annotate them so that this tool suite recognizes them (the "XS attribute"), then the file sorted before submitting to Cufflinks. Tophat does all of this by design for spliced reads. If you run the mapping jobs outside of Galaxy, be aware of how important the reference genome and any related annotation files (GFF, GTF) planned to be used downstream are to the success of the results. All inputs must be an /exact match/. These wiki sections explain: http://wiki.galaxyproject.org/Support#Reference_genomes http://wiki.galaxyproject.org/Support#Tools_on_the_Main_server http://wiki.galaxyproject.org/Support#Custom_reference_genome (best for smaller genomes, for larger genomes try a cloud Galaxy with scaled resources for best performance) Best, Jen Galaxy team On 10/16/13 12:56 AM, Thomas, Mervyn G. (Dr.) wrote:
Hi All, I am trying to analyse some human RNA-seq dataset which is in BAM format to generate FPKM values using cufflinks. The ensembl group have aligned the data using BWA 0.5.9 to generate these BAM datasets. I realise that TopHat alignments are optimal for Cufflinks, but is it possible for me to use cufflinks or some other tool to derive FPKM values from BWA aligned reads? I tried using cufflinks on one of the BAM files but got an error: Error running cufflinks. return code = -11 Command line: cufflinks -q --no-update-check -I 300000 -F 0.100000 -j 0.150000 -p 8 /galaxy-repl/main/files/006/853/dataset_6853120.dat The additional output was: cufflinks v2.1.1 cufflinks -q --no-update-check -I 300000 -F 0.100000 -j 0.150000 -p 8 I have sent it to the galaxy team and awaiting their reply. Any thoughts about how I can go about analysing this data. Best wishes Mervyn Dr. Mervyn G Thomas BSc, MBChB, PhD Academic Foundation Doctor School of Medicine University of Leicester UK
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Thomas, Mervyn G. (Dr.)