dear all,
I have a very tricky problem when using trim.seq on my Miseq 2x150 set of amplicons! I have assembled pair-end reads (consensus sequences), each sequence tagged with a combination of two different barcodes (one barcode in forward and another barcode in reverse), and the barcodes have not all the same length. Like this:
barcode AAGCCTTGCGTCAGACA TATCGACGATGT Sample 1
barcode AAGCCTTGCGTCAGACA CTCGATGATCACG Sample 2
barcode AAGCCTTGCGTCAGACA GCGATCAGCAGATC Sample 3
barcode AAGCCAAGGGAGGAGAC TATCGACGATGT Sample 4
barcode AAGCCAAGGGAGGAGAC CTCGATGATCACG Sample 5
barcode AAGCCAAGGGAGGAGAC GCGATCAGCAGATC Sample 6
when using trim.seq I am able to recover one part of samples (the first combination for each set of forward and reverse tag or about 50 000 sequences from a total of one billion), and not the rest. I reverse complemented the reverse tags but it does not works either. I do not visualize very well how trim.seq is working but I think that I am not telling it correctly how to read my tag combinations.
is there anyone who could help me with this. I am finishing my PhD very soon, I am a little stressed at this very moment.
thanks a lot in advance.
cheers,
stefaniya