Qiime and metabarcoding

[Angela.S]

HI, I am a new user of Qiime and am analyzing metabarcoding data run as single end reads on the Illumina Miseq. Our target gene primers have identical barcodes attached to both ends and groups of samples are pooled and indexed through ligation of TruSeq adaptors. These are demultiplexed off the instrument. My questions are as follows:

1. Since both orientations are possible, do I include both primer (5'-3') possibilities in the “LinkerPrimerSequence” column of the mapping file (e.g. xxxxxx,xxxxx) or does the 'split_libraries.py' do that automatically?

2. Do I include the barcode sequence in both the "BarcodeSequence" and “LinkerPrimerSequence” or just the target primer in the “LinkerPrimerSequence” column of the mapping file?

3.If I use the ‘truncate_remove’ function, do I also need both R possibilities (i.e. xxxxxx,xxxxxxx)?

I have tried the 'split_libraries.py' with two different mapping files and found that the one where I used both possibilities in “LinkerPrimerSequence” and “ReversePrimer” yielded ~ 2x the numbers in the split_library_log.txt. I just want to make sure this is correct?

Thanks,

Angela