Hope you’re all enjoying the weekend! I have a few QIIME2 questions - please forgive me if they are naïve/dumb – I’m very new to all of this. I just ran the denoising step with DADA2 and I’m worried that I have contaminants in my negative controls. I want to make sure that I used the right parameters in truncating and trimming before telling my PI we have contamination.
We are using a 534R / 27F primer combo (V1-V3 primer). From my research online, I took this to mean that our amplicon is 507 bp (534-507) long. With 300 bp forward and reverse sequences, this should give us 93 bp overlap (600-507). I read that we should aim for 20nt+natural variation in our target for overlap and that we should truncate where the median quality score drops below 20.
However, when I looked at our interactive quality plot (demux.qzv), our median quality score dropped below 20 at positions 263 for forward and 202 for the reverse, respectively
• 263+202-507 = -38, which means we get no overlap
• To get some overlap, I lowered the median quality score cutoff to the point where we could get some overlap and ended up running the following command:
• qiime dada2 denoise-paired
• --i-demultiplexed-seqs demux.qza
• --p-trunc-len-f 300
• --p-trunc-len-r 245
• --o-table table.qza
• --o-representative-sequences rep-seqs.qza
• --o-denoising-stats denoising-stats.qza
When I looked at the table.qzv output, I saw that our negative controls had sequence counts greater than 0 for negative controls (NC.Kit4, NC.Kit5. Someone in my lab told me that we should have sequence counts of 0 for the negative controls after denoising, otherwise, this means we have contamination.
My questions are:
- Am I using the right parameters for denoising?
- Do I have contamination in my negative controls?