Dear All,
I would like to add some filtering steps in my RNA-Seq pipeline. To do so,
I used the accepted.hits from TopHat and apply a filter using  NGS: SAM
Tools > Filter SAM and select reads with bitwise flag 0x0002. This does the
job. However, I am unable to use cufflink after this step and got the
following error message that seems to indicate that the file contains no
header and is unsorted. Is there a workaround ?
Thanks a lot

Error running cufflinks.
return code = 1
cufflinks: /lib64/ no version information available (required by
Command line:
cufflinks -q --no-update-check -s 20 -I 300000 -F 0.100000 -j 0.150000 -p 8
-m 200 -g /galaxy/main_pool/pool5/files/003/858/dataset_3858145.dat
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File /galaxy/main_pool/pool1/files/003/858/dataset_3858306.dat doesn't
appear to be a valid BAM file, trying SAM...
[14:11:28] Loading reference annotation.
[14:11:28] Inspecting reads and determining fragment length distribution.

Error: this SAM file doesn't appear to be correctly sorted!
        current hit is at chr10:181061, last one was at chr1:245006405
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.

Denis Puthier
laboratoire INSERM
Parc Scientifique de Luminy case 928
163, avenue de Luminy
13288 MARSEILLE cedex 09
Tel: (National) 04 91 82 87 11 / (International) 33 4 91 82 87 11
Fax: (National) 04 91 82 87 01 / (International) 33 4 91 82 87 01

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