Inadequate overlap for V3-V4 (paired-end) due to quality drop

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Hi everyone,

I am currently analyzing 16S rRNA gene sequences (V3-V4 region) using QIIME2. I am facing a challenge during the denoising step with dada2 denoise-paired.

The problem is that my sequencing quality drops significantly around 220 bp for the Fw reads and 190 bp for the Rv reads. Taking this approach my combined high-quality length would be 410 bp, so I would be approximately 70 bp short of achieving the required 20 bp overlap for merging.

So, my questions are:

  1. If I prioritize sequence quality and truncate at 220/190 bp, the reads will not overlap. Is there any reliable evidence or benchmark regarding the taxonomic resolution (specifically at the species level) when using non-overlapping paired-end reads (e.g., using N-padding or concatenation)?

  2. In this scenario, would you recommend proceeding with Fw reads only (denoise-single) to maintain high quality, or is it better to risk lower quality bases to force an overlap?

  3. Are there any alternative strategies within QIIME 2 to handle V3-V4 samples with poor distal quality without losing too much taxonomic depth?

Thanks in advance for the help!

Gorka

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