Phred score cutoff vs. sequence length for an unbiased comparison between different cohorts

[Nicholas_Bokulich]

Hi @Parix,
Great questions!

@Parix

different organisms which vary in sequencing quality considering Phred score plots.

I am assuming these are from different sequencing runs?

@Parix

I was wondering if I should choose a specific Phred score for all OR have the same trim and truncate parameters so that the resulting sequences would have comparable lengths?

The most important thing is that the sequence lengths are the same if you plan to use denoising methods to denoise and dereplicate. If sequences are not the same length, each run will have its own unique ASVs and you would have trouble comparing them.

So if the reads are single-end it will be important to trim them at the same lengths. If they are paired end, then theoretically the merged reads should yield the same length and same amplicons.

Another way to handle this if you must trim to different lengths is to use closed-reference OTU clustering to cluster the reads against known full-length reference sequences. It's a lazy but efficient way to solve what is sometimes an intractable problem.

@Parix

The same issue exists regarding sequencing depths which vary between the studied organisms. In this case, should the rarefaction depth be specific to each studied animal?

You should rarefy all to the same depth if you plan to compare these for alpha or beta diversity analyses. Otherwise it would be like s and s!

Good luck!