reads.fa ids do not have the correct format it must have the id line >SSS_INT_xINT SSS is a three letter code indicating the sample origin INT is just a running number xINT is the number of read occurrences You can use the mapper.pl module to create such a file from a fasta file with mapper.pl reads.fa -c -m -s reads.fa.collapsed See also the mapper.pl help for more information on preprocessing input files.