I had finally found a workaround by selecting header line matching ^@ and merging this with the SAM file. but I think your solution is far more elegant. I'll try.

Thanks

2012/2/28 Carlos Borroto <carlos.borroto@gmail.com>

Hi Denis,

In a similar situation I was able to move forward using NGS: Picard
(beta) / Replace SAM/BAM Header to copy back the header from the
original unfiltered BAM or SAM file.

Hope it helps,
Carlos

On Tue, Feb 28, 2012 at 3:15 PM, denis puthier <puthier@tagc.univ-mrs.fr> wrote:
> 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
>
> http://main.g2.bx.psu.edu/u/dputhier/h/srx011549
>
>
> Error running cufflinks.
> return code = 1
> cufflinks: /lib64/libz.so.1: no version information available (required by
> cufflinks)
> 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
> /galaxy/main_pool/pool1/files/003/858/dataset_3858306.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
> TAGC/INSERM U928
> Parc Scientifique de Luminy case 928
> 163, avenue de Luminy
> 13288 MARSEILLE cedex 09
> FRANCE
> Mail: puthier@tagc.univ-mrs.fr
> 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
>
> Web:
> http://tagc.univ-mrs.fr/puthier
> http://biologie.univ-mrs.fr/view-data.php?id=245
> http://tagc.univ-mrs.fr/tbrowser
> ====================================================================
>

> ___________________________________________________________
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___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
at usegalaxy.org. Please keep all replies on the list by
using "reply all" in your mail client. For discussion of
local Galaxy instances and the Galaxy source code, please
use the Galaxy Development list:

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--
====================================================================
Denis Puthier
laboratoire INSERM
TAGC/INSERM U928
Parc Scientifique de Luminy case 928
163, avenue de Luminy
13288 MARSEILLE cedex 09
FRANCE
Mail: puthier@tagc.univ-mrs.fr
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

Web:
http://tagc.univ-mrs.fr/puthier
http://biologie.univ-mrs.fr/view-data.php?id=245
http://tagc.univ-mrs.fr/tbrowser
====================================================================