Questions about ITS Region Amplicon Sequencing Quality Using Illumina MiSeq

[ksv6178]

Hi, all.

I'm analyzing fungal ITS amplicon data from gut samples. However, the data quality isn't very good—there's a noticeable drop in quality after around 240–250 bp.

Additionally, when I researched other ITS amplicon data, I found some datasets with similarly low quality.

So, I wanted to ask: If you’ve worked with ITS amplicon data, have you experienced issues with low-quality reads?

Thank you!

[ovon]

I have, and that drop in quality after 250bp isn't unexpected in most amplicons. Using the dada2 ITS-specific protocol should be helpful, in that you can trim reads while accounting for length variability of the amplicon. I find that using this protocol and being more permissive about maxEE in dada2 - which the software is capable of handling safely - allows reasonable and even good read retention (average of >70% of reads retained) even in samples where read quality plots look a bit dubious

Protocol is here: https://benjjneb.github.io/dada2/ITS_workflow.html