Poor results, please I need support

User Support
2

Hi @leandro_di_gloria welcome to :qiime2:!

I think you'll greatly improve your results if you simply add the following flag to your command: --p-match-adapter-wildcards. Otherwise the ambiguous IUPAC bases like BWN... will count as mismatches and increase the chances of being unable to find and trim the primers.

Given that you are providing the primers in the normal and reverse complimented form, I assume you are expecting your sequence data in mixed orientation? As in this case:

Here is the updated command with other minor modifications.. note that I removed the ' and the ...:

qiime cutadapt trim-paired \
    --i-demultiplexed-sequences demux-paired-end.qza \
    --p-adapter-f  CCTACGGGNGGCWGCAG  GGATTAGAWACCCBNGTAGTC  \
    --p-adapter-r GACTACNVGGGTWTCTAATCC  CTGCWGCCNCCCGTAGG \
    --p-cores 4 \
    --o-trimmed-sequences trimmed-seqs.qza \
    --p-discard-untrimmed \
    --p-match-adapter-wildcards \
    --verbose

Note if your reads are in mixed orientation you'll want orient your reads to the same direction using another tool, like the RESCRIPt plugin rescript orient-seqs command.

If you are not expecting reads in mixed orientation you can run the command like so:

qiime cutadapt trim-paired \
    --i-demultiplexed-sequences demux-paired-end.qza \
    --p-adapter-f  CCTACGGGNGGCWGCAG  \
    --p-adapter-r GACTACNVGGGTWTCTAATCC  \
    --p-cores 4 \
    --o-trimmed-sequences trimmed-seqs.qza \
    --p-discard-untrimmed \
    --p-match-adapter-wildcards \
    --verbose

Keep us informed. :slight_smile:
-Mike