RNA seq data analysis-Cuffmerge problem

[titua19810]

Hi Everyone,

I am new to RNA seq data and trying to learn tophat protocols using example data. I am at the stem p 4 of nature protocol paper (Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks)
as in step 4 of the nature protocol my code follows (shown below) and the run was complete without any error message but lot of warning message (please see below) as well as following message
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File ./merged_asm/tmp/mergeSam_fileVb2mYW doesn't appear to be a valid BAM file, trying SAM...
[16:00:25] Loading reference annotation.

Now my question is whether the run was successfully completed or not. I would greatly appreciate your input about this issue. Thanks for your help




[Tue Jun 4 16:00:20 2013] Preparing output location ./merged_asm/
[Tue Jun 4 16:00:23 2013] Converting GTF files to SAM
[16:00:23] Loading reference annotation.
[16:00:24] Loading reference annotation.
[Tue Jun 4 16:00:25 2013] Quantitating transcripts
You are using Cufflinks v2.1.1, which is the most recent release.
Command line:
cufflinks -o ./merged_asm/ -F 0.05 -g /u/scratch/a/arkhan/Mus_musculus/UCSC/mm9/Annotation/Genes/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_fileVb2mYW
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File ./merged_asm/tmp/mergeSam_fileVb2mYW doesn't appear to be a valid BAM file, trying SAM...
[16:00:25] Loading reference annotation.
[16:00:29] Inspecting reads and determining fragment length distribution.
Processed 21449 loci.
> Map Properties:
> Normalized Map Mass: 22931.00
> Raw Map Mass: 22931.00
> Fragment Length Distribution: Truncated Gaussian (default)
> Default Mean: 200
> Default Std Dev: 80
[16:00:30] Assembling transcripts and estimating abundances.
Processed 21449 loci.
[Tue Jun 4 16:01:11 2013] Comparing against reference file /u/scratch/a/arkhan/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf
You are using Cufflinks v2.1.1, which is the most recent release.
No fasta index found for /u/scratch/a/arkhan/Mus_musculus/UCSC/mm9/Sequence/Bowtie2Index/genome.fa. Rebuilding, please wait..
Fasta index rebuilt.
Warning: couldn't find fasta record for 'chr13_random'!
Warning: couldn't find fasta record for 'chr17_random'!
Warning: couldn't find fasta record for 'chr1_random'!
Warning: couldn't find fasta record for 'chr4_random'!
Warning: couldn't find fasta record for 'chr5_random'!
Warning: couldn't find fasta record for 'chr7_random'!
Warning: couldn't find fasta record for 'chr8_random'!
Warning: couldn't find fasta record for 'chr9_random'!
Warning: couldn't find fasta record for 'chrUn_random'!
Warning: couldn't find fasta record for 'chrX_random'!
Warning: couldn't find fasta record for 'chrY_random'!
[Tue Jun 4 16:02:30 2013] Comparing against reference file /u/scratch/a/arkhan/Mus_musculus/UCSC/mm9/Annotation/Genes/genes.gtf
You are using Cufflinks v2.1.1, which is the most recent release.
Warning: couldn't find fasta record for 'chr13_random'!
Warning: couldn't find fasta record for 'chr17_random'!
Warning: couldn't find fasta record for 'chr1_random'!
Warning: couldn't find fasta record for 'chr4_random'!
Warning: couldn't find fasta record for 'chr5_random'!
Warning: couldn't find fasta record for 'chr7_random'!
Warning: couldn't find fasta record for 'chr8_random'!
Warning: couldn't find fasta record for 'chr9_random'!
Warning: couldn't find fasta record for 'chrUn_random'!
Warning: couldn't find fasta record for 'chrX_random'!
Warning: couldn't find fasta record for 'chrY_random'!