I have a question regarding the use of an oligos file in the make.contigs step. So far in my amplicon analysis, I have not removed the primer sequences (including the barcode in the reverse primer) in the beginning of the pipeline, due to the assumption that these will be trimmed away during alignment against the Silva 16S database.
I suspect I have quite many «raw» reads with >1 mismatch to the barcode sequence, and now I want those to not be assembled during «make.contigs». As I understand, it’s possible to provide the primer and barcode sequences in an «oligos file» in this command, and specify the amount of allowed mismatches (I want 0 to the barcodes) to these sequences. I use the Caporaso primers (used by Earth Microbiome Project), which have a 12 bp Golay barcode in the reverse primer, and also spacer and linker (see ftp://ftp.metagenomics.anl.gov/data/misc/EMP/SupplementaryFile1_barcoded_primers_515F_806R.txt). My oligos file is therefore like shown below (tab separated). When used, I get no assembled contigs at all. When not used, I get a lot of contigs assembled.
Why isn’t my oligos file working in the “make.contigs” step?
primer GTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT V4
barcode none GGAGACAAGGGA L0814_25G1
barcode none AATCAGTCTCGT L0814_25G2
barcode none AATCCGTACAGC L0814_25G3
barcode none CCAGTGTATGCA L0814_27G1
barcode none CCTCGTTCGACT L0814_27G2
barcode none TGAGTCACTGGT L0814_27G3
barcode none CACGCCATAATG L0814_28G1
barcode none CAGGCGTATTGG L0814_28G2
barcode none GGATCGCAGATC L0814_28G3