Cutadapt seems to be discarding too many reads (~60%). We suspect this has to do with the mixed orientation flag. We have also spot checked a few reads from the demultiplexed file and there are some reads in each orientation. We have paired end sequencing with the same barcodes on each end. Removing the mixed orientation flag did not change the number of outputted reads. Any tips to improve this?
See example output from demux-paired command:
CGGCTYAATTYGAYTCAACRC (forward primer)
CGGCTTAATTTGACTCAACGCGGGGAAGCTCACCCGGCCCGGACACGGAAAGGATTGACAGATTGATAGCTCTTTCTCGATTCTGTGGGTGGTGGTGCATGGCCGTTCTTAGTTGGTGGAGTGATTTGTCTGGTTGATTCCGATAACGAACGAGACTCCGGCTTGTTAACTAGTTTCGCGGCCCCGTGTGGTCGGCGACTAACTTCTTAGAGGGACAAGTGGCGTTCAGCCACACGAGAAGGAGC
+
GGGCATCACRGACCTG (Reverse complement of reverse primer)
GGGCATCACAGACCTGTTATTGCTCCTTCTCGTGTGGCTGAACGCCACTTGTCCCTCTAAGAAGTTAGTCGCCGACCACACGGGGCCGCGAAACTAGTTAACAAGCCGGAGTCTCGTTCGTTATCGGAGTCAACCAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACCACCCACAGAATCGAGAAAGAGCTATCAATCTGTCAATCCTTTCCGTGTCCGGGCCGGGTGAGCTTCCCC