cuffdiff error, issues with bam sorting

[rhcr56]

Hello,

I have used the tuxedo suite in the past and never run into this issue. I checked the headers for the alignment files (bam) and they are lexicographically sorted. Furthermore, the chromosomes in the merged.gtf file that I'm using is sorted identically. Yet, when I run cuffdiff like this

/ircf/ircfapps/bin/cuffdiff -p 4 -o ./Cuffdiff/ -L E14,E16,E18,E20,P1,P7 --total-hits-norm -b rn4.fa -u -c 10 --FDR 0.01 merged.gtf accepted_hits1.bam,accepted_hits2.bam accepted_hits3.bam,accepted_hits4.bam accepted_hits5.bam,accepted_h
its6.bam accepted_hits7.bam,accepted_hits8.bam accepted_hits9.bam,accepted_hits10.bam accepted_hits11.bam,accepted_hits12.bam

i get this error

You are using Cufflinks v2.0.2, which is the most recent release.
[13:56:25] Loading reference annotation and sequence.
[13:56:56] Inspecting maps and determining fragment length distributions.

Error: this SAM file doesn't appear to be correctly sorted!
current hit is at chr1:589946, last one was at chr1:615546
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.

Additionally, I tried to resort the bam files, and samtools gave me this error

[bam_sort_core] truncated file. Continue anyway.

I'm stuck. This wasn't an issue six months ago. I've looked on all other forums and tried their suggestions and got nowhere. Thanks.

[swbarnes2]

Hello,

I have used the tuxedo suite in the past and never run into this issue. I checked the headers for the alignment files (bam) and they are lexicographically sorted. Furthermore, the chromosomes in the merged.gtf file that I'm using is sorted identically. Yet, when I run cuffdiff like this

/ircf/ircfapps/bin/cuffdiff -p 4 -o ./Cuffdiff/ -L E14,E16,E18,E20,P1,P7 --total-hits-norm -b rn4.fa -u -c 10 --FDR 0.01 merged.gtf accepted_hits1.bam,accepted_hits2.bam accepted_hits3.bam,accepted_hits4.bam accepted_hits5.bam,accepted_h
its6.bam accepted_hits7.bam,accepted_hits8.bam accepted_hits9.bam,accepted_hits10.bam accepted_hits11.bam,accepted_hits12.bam

i get this error

You are using Cufflinks v2.0.2, which is the most recent release.
[13:56:25] Loading reference annotation and sequence.
[13:56:56] Inspecting maps and determining fragment length distributions.

Error: this SAM file doesn't appear to be correctly sorted!
current hit is at chr1:589946, last one was at chr1:615546
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.

Additionally, I tried to resort the bam files, and samtools gave me this error

[bam_sort_core] truncated file. Continue anyway.

I'm stuck. This wasn't an issue six months ago. I've looked on all other forums and tried their suggestions and got nowhere. Thanks.

Try using Picard to sort them. Picard, unlike, say, samtools, will put in the header that the file is sorted. Usually, you want these things sorted by coordinate.