Hi,
I have been using tophat and cufflinks to analyse some RNA seq data, but cuffmerge is not behaving as I would expect.
For all steps of the analysis I have used a reference gff3 file for RABT.
For tophat I specifed:
and on subsequent usage just
For cufflinks I specified
All the fpkm_tracking and gtf output files contain annotations from the original gtf file, including from transcripts with very high coverage so not just faux reads.
When using cuffmerge for the 16 samples I use the following command:
In none of the output files is there any mention of the annotations from the gff file.
I thought that the problem was that it is a gff file and not a gtf file, so I converted using
However the output is the same. There is also no mention of the gtf file in the log file for either gff3 or gtf:
And nothing is prefixed / goes to the folder cuffmerge that i specifed with the -o flag, it only goes to the merged_asm folder.
Can anyone tell me what I am doing wrong or if this behaviour is as expected?
Version is: cufflinks v2.0.2
Many Thanks
Anna
I have been using tophat and cufflinks to analyse some RNA seq data, but cuffmerge is not behaving as I would expect.
For all steps of the analysis I have used a reference gff3 file for RABT.
For tophat I specifed:
Code:
-G <gff file> --transcriptome-index <transcriptome prefix>
Code:
--transcriptome-index <transcriptome prefix>
Code:
--GTF-guide <gff file>
When using cuffmerge for the 16 samples I use the following command:
Code:
cuffmerge <cufflinks gtf list file> -o cuffmerge -g <gff file> -p 17
I thought that the problem was that it is a gff file and not a gtf file, so I converted using
Code:
gffread <gff file> -T -o <gtf file>
Code:
cuffmerge <cufflinks gtf list file> -o cuffmerge -g <gtf file> -p 17
Code:
/software/additional/bin/cuffmerge <path>/sam/tophat/cufflinks/gtfFileList.txt -o cuffmerge -g <path>/data/abH97_filt.gffread.gtf -p 17 gtf_to_sam -F <path>/sam/tophat/1_1/cufflinks/transcripts.1_1.gtf ./merged_asm/tmp/gtf2sam_filenEmb9h gtf_to_sam -F <path>/sam/tophat/1_2/cufflinks/transcripts.1_2.gtf ./merged_asm/tmp/gtf2sam_fileeDtnvd gtf_to_sam -F <path>/sam/tophat/1307_M5/cufflinks/transcripts.1307_M5.gtf ./merged_asm/tmp/gtf2sam_fileQhIpNd gtf_to_sam -F <path>/sam/tophat/1_3/cufflinks/transcripts.1_3.gtf ./merged_asm/tmp/gtf2sam_fileAHuzXi gtf_to_sam -F <path>/sam/tophat/2_1/cufflinks/transcripts.2_1.gtf ./merged_asm/tmp/gtf2sam_fileZw6YGs gtf_to_sam -F <path>/sam/tophat/2_2/cufflinks/transcripts.2_2.gtf ./merged_asm/tmp/gtf2sam_file87N56G gtf_to_sam -F <path>/sam/tophat/2_3/cufflinks/transcripts.2_3.gtf ./merged_asm/tmp/gtf2sam_file6ipSj0 gtf_to_sam -F <path>/sam/tophat/A151_M5/cufflinks/transcripts.A151_M5.gtf ./merged_asm/tmp/gtf2sam_file3SWsto gtf_to_sam -F <path>/sam/tophat/F25_2_1/cufflinks/transcripts.F25_2_1.gtf ./merged_asm/tmp/gtf2sam_fileC2SotR gtf_to_sam -F <path>/sam/tophat/F25_2_3/cufflinks/transcripts.F25_2_3.gtf ./merged_asm/tmp/gtf2sam_filelpirkp gtf_to_sam -F <path>/sam/tophat/F25_2_4/cufflinks/transcripts.F25_2_4.gtf ./merged_asm/tmp/gtf2sam_fileJX9sR1 gtf_to_sam -F <path>/sam/tophat/F25_2_M5/cufflinks/transcripts.F25_2_M5.gtf ./merged_asm/tmp/gtf2sam_filezkS48I gtf_to_sam -F <path>/sam/tophat/F25_2_M6/cufflinks/transcripts.F25_2_M6.gtf ./merged_asm/tmp/gtf2sam_file68Mb8u gtf_to_sam -F <path>/sam/tophat/I142_4/cufflinks/transcripts.I142_4.gtf ./merged_asm/tmp/gtf2sam_file6CwbQl gtf_to_sam -F <path>/sam/tophat/I142_M10/cufflinks/transcripts.I142_M10.gtf ./merged_asm/tmp/gtf2sam_fileeikjVg gtf_to_sam -F <path>/sam/tophat/I142_M9/cufflinks/transcripts.I142_M9.gtf ./merged_asm/tmp/gtf2sam_filetTRVKg sort -k 3,3 -k 4,4n --temporary-directory=./merged_asm//meta_asm_tmp/ ./merged_asm/tmp/gtf2sam_filenEmb9h ./merged_asm/tmp/gtf2sam_fileeDtnvd ./merged_asm/tmp/gtf2sam_fileQhIpNd ./merged_asm/tmp/gtf2sam_fileAHuzXi ./merged_asm/tmp/gtf2sam_fileZw6YGs ./merged_asm/tmp/gtf2sam_file87N56G ./merged_asm/tmp/gtf2sam_file6ipSj0 ./merged_asm/tmp/gtf2sam_file3SWsto ./merged_asm/tmp/gtf2sam_fileC2SotR ./merged_asm/tmp/gtf2sam_filelpirkp ./merged_asm/tmp/gtf2sam_fileJX9sR1 ./merged_asm/tmp/gtf2sam_filezkS48I ./merged_asm/tmp/gtf2sam_file68Mb8u ./merged_asm/tmp/gtf2sam_file6CwbQl ./merged_asm/tmp/gtf2sam_fileeikjVg ./merged_asm/tmp/gtf2sam_filetTRVKg > ./merged_asm/tmp/mergeSam_file1gh4tl cufflinks -o ./merged_asm/ -F 0.05 -q --overhang-tolerance 200 --library-type=transfrags -A 0.0 --min-frags-per-transfrag 0 --no-5-extend -p 1 ./merged_asm/tmp/mergeSam_file1gh4tl cuffcompare -o tmp_meta_asm -C -G ./merged_asm//transcripts.gtf cuffcompare -o tmp_meta_asm -C -G ./merged_asm//merged.gtf
Can anyone tell me what I am doing wrong or if this behaviour is as expected?
Version is: cufflinks v2.0.2
Many Thanks
Anna