Originally posted by ndaniel
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The SAM file supplied to Cufflinks must be sorted by reference position. If you aligned your reads with TopHat, your alignments will be properly sorted already. If you used another tool, you may want to make sure they are properly sorted as follows:
sort -k 3,3 -k 4,4n hits.sam > hits.sam.sorted
And you may also have to filter the reads not mapped to the reference genome, marked by "*" in the field of RNAME.
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