After running cufflink for two samples of RNA-Seq data, I used cuffmerge and executed the following command
cuffmerge -o -g -s assemblies.txt
Where assemblies.txt contains the transcripts.gtf
But I am getting following error
Beginning transcriptome assembly merge
Preparing output location cuffmerge_out/ Converting GTF files to SAM Loading reference annotation. Loading reference annotation. 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 cuffmerge_out/ -F 0.05 -g sequence.gtf -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 1 cuffmerge_out/tmp/mergeSam_filelA9PHi [bam_header_read] EOF marker is absent. [bam_header_read] invalid BAM binary header (this is not a BAM file). File cuffmerge_out/tmp/mergeSam_filelA9PHi doesn't appear to be a valid BAM file, trying SAM... Loading reference annotation. Inspecting reads and determining fragment length distribution.
Error: this SAM file doesn't appear to be correctly sorted! current hit is at U42361.1:174, last one was at U44758.1:645 Cufflinks requires that if your file has SQ records in the SAM header that they appear in the same order as the chromosomes names in the alignments. If there are no SQ records in the header, or if the header is missing, the alignments must be sorted lexicographically by chromsome name and by position.
[FAILED] Error: could not execute cufflinks
I think the first thing you need to do is sort the sam files. You can use samtools to do this. The documentation is here: http://www.htslib.org/doc/samtools.html
Sophie Sneddon I think not. Since she is using GTF file as input which is converted to sam file, its the gtf file which is not formatted correctly or the reference miss some information.
It seems that the tool is looking for a sorted SAM/BAM file with headers. Could you check if your cufflinks output folder contains sorted alignment files with the same names and in same order as specified in the list of GTF files - i.e. assemblies.txt ? It's a bit odd because it seems like Cuffmerge only requires the list of GTF files with full paths. I am not sure why it searches for the sorted SAM, but just check if they exist at the same path. Another suggestion is to use StringTie instead of Cufflinks. StringTie is from the same team and is much faster and supposedly more accurate than Cufflinks https://ccb.jhu.edu/software/stringtie/index.shtml?t=faq
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