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Old 04-05-2012, 05:43 AM   #1
crader
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Location: Cardiff, UK

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Default cuffdiff says SAM isnít sorted, although it handled it cufflinks

Hi all, I'm having an issue with cufflinks in that I can get through cufflinks with a de novo, but am halted here:

cuffdiff -o diff_out -b annolrubtran.fasta -p 20 -L 0mg,36mg,125mg -u cuffcmp.combined.gtf ./tophat008paired/accepted_hits.sam.sorted ./tophat009paired/accepted_hits.sam.sorted ./tophat010paired/accepted_hits.sam.sorted
You are using Cufflinks v1.3.0, which is the most recent release.
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File ./tophat008paired/accepted_hits.sam.sorted doesn't appear to be a valid BAM file, trying SAM...
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File ./tophat009paired/accepted_hits.sam.sorted doesn't appear to be a valid BAM file, trying SAM...
[bam_header_read] EOF marker is absent.
[bam_header_read] invalid BAM binary header (this is not a BAM file).
File ./tophat010paired/accepted_hits.sam.sorted doesn't appear to be a valid BAM file, trying SAM...
[19:24:38] Loading reference annotation and sequence.
[19:25:40] Inspecting maps and determining fragment length distributions.

Error: this SAM file doesn't appear to be correctly sorted!
current hit is at Contig10010|nucleolin-:26, last one was at Contig1000|---NA---:198
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.


I've prviouisly converted the tophat output into a .sam before sorting:

samtools view -h -o accepted_hits.sam accepted_hits.bam
sort -k 3,3 -k 4,4n accepted_hits.sam > accepted_hits.sam.sorted

This feeds into cufflinks alright, but stops as above when shuttled into cuffdiff. I also used cuffcompare to get the combined.gtf- If anyone could offer me a suggestion, I'd thoroughly appreciate it!!
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Old 04-05-2012, 07:56 AM   #2
crader
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Update (I've also posted on Biostar)

It starts to give me hassles as here: http://biostar.stackexchange.com/que...into-cuffmerge when I put the .bam files in. Is there a faster work-around without changing the hitts.cpp? (I don't have permissions and am working off site).
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Old 04-05-2012, 08:43 AM   #3
ETHANol
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There was a thread about this the other day and the problem was with chrM.
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