Losing reads at DADA2 Merge step 30% of my samples lost over 50% - I have tried multiple steps to fix- please help

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1

Hello,

I am running fish skin swabs for 16s rRNA 515F/806R sequences using Illumina MiSeq, V2, 500 cycle kit (2x250).

I am having an issue with the merging step in DADA2. Around 30% of my samples have 50% loss at the merging step. I have tried to truncate at different lengths, and it all results in the same outcome. Furthermore, the quality plots are pretty good! I have already used cut adapt to remove the primers. If there is anything else someone can think of that would be helpful. I am currently thinking that there most be a lot of issues with the actually sequences that isn't showing on the quality plots causing the forward and reverse reads to be different and fail at merging.

#tried p-trunc-len-f 200, 220, 230, 170
#tried p-trunc-len-r 120,210, 220, 170

qiime dada2 denoise-paired \
  --i-demultiplexed-seqs paired-demux-16S-adapter-trim.qza \
  --p-trunc-len-f 200 \ 
  --p-trunc-len-r 120 \ 
  --o-representative-sequences rep-seqs.qza \
  --o-table table.qza \
  --o-denoising-stats denoising-stats.qza \
  --p-n-threads 16 \
  --verbose

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2

Hello and welcome to the forum!

Thank you for providing all the necessary details - it makes it easier to understand the issue!
It looks like you are losing most of the reads at the merging step, despite trying several truncation sets. Since the V4 region is pretty short, I doubt that your reads are not overlapping.

I suspect that the main issue is the quality scores. Could you try:

  • set higher max-ee values for F and r reads
  • decrease the minimum overlap between reads (just in case)

The good news is that, as I wrote above, the V4 region is pretty short and your forward reads cover most of it even without reverse reads. In case nothing works, you should be fine to proceed with forward reads only.

Best,