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 diffout -b annolrubtran.fasta -p 20 -L 0mg,36mg,125mg -u cuffcmp.combined.gtf ./tophat008paired/acceptedhits.sam.sorted ./tophat009paired/acceptedhits.sam.sorted ./tophat010paired/acceptedhits.sam.sorted You are using Cufflinks v1.3.0, which is the most recent release. [bamheaderread] EOF marker is absent. [bamheaderread] invalid BAM binary header (this is not a BAM file). File ./tophat008paired/acceptedhits.sam.sorted doesn't appear to be a valid BAM file, trying SAM... [bamheaderread] EOF marker is absent. [bamheaderread] invalid BAM binary header (this is not a BAM file). File ./tophat009paired/acceptedhits.sam.sorted doesn't appear to be a valid BAM file, trying SAM... [bamheaderread] EOF marker is absent. [bamheaderread] 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 acceptedhits.sam acceptedhits.bam

sort -k 3,3 -k 4,4n acceptedhits.sam > acceptedhits.sam.sorted

This feeds into cufflinks alright, but stops as above when shuttled into cuffdiff. If anyone could offer me a suggestion, I'd thoroughly appreciate it!!