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.