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Cuffdiff sort error

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  • Cuffdiff sort error

    Hi All,
    I'm new to RNA-Seq and am trying to determine differential expression from a de Novo transcriptome assembly.

    I've constructed the transcriptome with velvet/oases, mapped the reads back to the transcriptome with bowtie, run cufflinks on the mapped reads, merged the .gtf files with cuffmerge and then looked for DE with cuffdiff. Unfortunately when I run cuffdiff I get the following error message:

    [13:49:30] Loading reference annotation.
    [13:50:01] Inspecting maps and determining fragment length distributions.

    Error: this SAM file doesn't appear to be correctly sorted!
    current hit is at Locus_10006_Transcript_1/6_Confidence_3.000_Length_452:87, last one was at Locus_10004_Transcript_7/17_Confidence_10.000_Length_594:156
    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.

    Frustratingly, the SAM file is the same sorted file I used as input for cufflinks. So why is it now a problem for cuffdiff? There appear to be no SQ records in the header and I've tried many different methods of resorting.

    Where have I gone wrong?

  • #2
    Have you tried using the BAM files instead of the sorted SAM files. I settled mine using the BAM files