Hi There, I am newbie to RNAseq analysis with basic knowledge of linux and perl. I am using tutorial from "Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks---by Cole tarpnell" protocol to run a trial run so as to get familiarized with data analysis process. I am using exact files mentioned in the protocol and have managed to generate required files after running tophat and cufflinks. Now I am trying to cuffmerge of files using following command. :cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt I have tried this several times and everytime end up with following error. Can anyone help me with this?
================================================================================================List of files C1R1clout/ C1R3thout/ C2R3clout/ genome.2.ebwt@ genome.rev.2.ebwt@ GSM794485C1R31.fq GSM794487C2R22.fq
C1R1thout/ C2R1clout/ C2R3thout/ genome.3.ebwt@ GSM794483C1R11.fq GSM794485C1R32.fq GSM794488C2R3_1.fq
C1R2clout/ C2R1thout/ Drosophilamelanogaster/ genome.4.ebwt@ GSM794483C1R12.fq GSM794486C2R11.fq GSM794488C2R32.fq
C1R2thout/ C2R2clout/ genes.gtf@ genome.fa@ GSM794484C1R21.fq GSM794486C2R12.fq README.txt*
C1R3clout/ C2R2thout/ genome.1.ebwt@ genome.rev.1.ebwt@ GSM794484C1R22.fq GSM794487C2R21.fq ussd-ftp.illumina.com/
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ayush(28)cuffmerge -g genes.gtf -s genome.fa -p 8 assemblies.txt
ERROR
[Fri Sep 21 12:17:13 2012] Beginning transcriptome assembly merge
[Fri Sep 21 12:17:13 2012] Preparing output location ./merged_asm/
[Fri Sep 21 12:17:15 2012] Converting GTF files to SAM
[12:17:16] Loading reference annotation.
[12:17:16] Loading reference annotation.
[12:17:17] Loading reference annotation.
[12:17:17] Loading reference annotation.
[12:17:18] Loading reference annotation.
[12:17:18] Loading reference annotation.
[Fri Sep 21 12:17:19 2012] Quantitating transcripts
You are using Cufflinks v2.0.2, which is the most recent release.
Command line:
cufflinks -o ./mergedasm/ -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 ./mergedasm/tmp/mergeSam_filekDEfQu
[bamheaderread] EOF marker is absent.
[bamheaderread] invalid BAM binary header (this is not a BAM file).
File ./mergedasm/tmp/mergeSamfilekDEfQu doesn't appear to be a valid BAM file, trying SAM...
[12:17:19] Loading reference annotation.
[12:17:20] Inspecting reads and determining fragment length distribution.
Processed 11271 loci.
Map Properties:
Normalized Map Mass: 66129.00
Raw Map Mass: 66129.00
Fragment Length Distribution: Truncated Gaussian (default)
Default Mean: 200 Default Std Dev: 80
[12:17:21] Assembling transcripts and estimating abundances.
Processed 11271 loci.
[Fri Sep 21 12:20:17 2012] Comparing against reference file genes.gtf
You are using Cufflinks v2.0.2, which is the most recent release.
No fasta index found for genome.fa. Rebuilding, please wait..
Fasta index rebuilt.
Warning: couldn't find fasta record for '2LHet'!
Warning: couldn't find fasta record for '2RHet'!
Warning: couldn't find fasta record for '3LHet'!
Warning: couldn't find fasta record for '3RHet'!
Warning: couldn't find fasta record for 'U'!
Warning: couldn't find fasta record for 'XHet'!
Warning: couldn't find fasta record for 'YHet'!
Warning: couldn't find fasta record for 'dmelmitochondriongenome'!
[Fri Sep 21 12:20:24 2012] Comparing against reference file genes.gtf
You are using Cufflinks v2.0.2, which is the most recent release.
Warning: couldn't find fasta record for '2LHet'!
Warning: couldn't find fasta record for '2RHet'!
Warning: couldn't find fasta record for '3LHet'!
Warning: couldn't find fasta record for '3RHet'!
Warning: couldn't find fasta record for 'U'!
Warning: couldn't find fasta record for 'XHet'!
Warning: couldn't find fasta record for 'YHet'!
Warning: couldn't find fasta record for 'dmelmitochondriongenome'!
