Hi all, I encountered the same problems. But the error message "mate did not have refenence tag" could only be removed by using the exact fastq header as provided in the example data files. This means I had to convert my headers from e.g.: @UNC10-SN254:517:H8PAKADXX:2:1101:1109:2033 1:N:0:TAGCTT to: @UNC10-SN254:517:H8PAKADXX:2:1101:1109:2033#TAGCTT/1 This solved the issue with the mate reference tag for me.