understanding the trade-off between dada2 single vs paired ends


I am working on Microbial Diversity on human skin using the V3-V4 300bp amplicon by Illumina.
All in all, I performed five independent sequencing runs. Especially on the first run, the reverse reads are of very poor quality. Even if I push out the truncations limits to a very low level I only get 1% of merged reads when using dada2-denoise-paired. Hence I decided to use dada2-denoise-single-reads.

1.) What kind/ amount of information am I losing if I use denoise-single instead of paired?

2.) Can I use denoise-single on the first run and denoise-paired on the later runs (where reverse reads look better) and later merge the table? Or will this result in a bias in downstream analyses?

Thanks very much in advance!


Hi @e_flat_minor ,

Sounds like an all-too-familiar problem :grin:

You will lose some amount of taxonomic resolution and ASV resolution (impacting diversity, etc), but probably not an enormous amount.

If you want to run a test to see what kind of information you lose, you could follow this tutorial but trimming with two different primer sets (V3 only vs. V3-V4).

This will certainly lead to biases, as the paired-end reads will be longer and somewhat taxonomically different from the single-end reads. If using ASV (denoising) methods, your ASVs will all have different IDs and so no ASVs will be shared between the single and paired-end runs!

On the other hand, if you do want to follow an approach like this you could use closed-reference OTU clustering to map all of the single- and paired-end reads against full-length reference sequences, then it will be possible to compare across runs. But then you lose other information (e.g., ASV-level variation, reads that do not map to a reference) so it is still a trade-off.

All in all, I'd suggest seeing what the single-end reads get you in terms of diversity and taxonomy results, then circle back if you think that you are losing too much.

Good luck!


Hi @Nicholas_Bokulich

Thank you very much for your helpful answer and clarification! I think I'll stick to dada2-denoise-single for now and maybe look at my sample processing/ PCR conditions for next experiments.

Best regards,

1 Like

From my point of view, this thread may be closed.

Thanks very much!

This topic was automatically closed 31 days after the last reply. New replies are no longer allowed.