Hello everyone!

We plan to perform 16S metabarcoding on groundwater samples, using the Illumina Iseq100 sequencer.

I was wondering which region and primers to use. I would like to perform 2x150 pb rather than 1x300 pb sequencing, to avoid bad quality reads at the 3' end (but not sure, if you have an opinion on that, fell free!).

So far, I was said to use the following primers:

1) 341F and 805R, as described here in the Illumina iseq100 application note (available here:https://support.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/iseq100-16s-app-note-770-2018-009.pdf)

But if you look closer, those primers target the V3-V4 region, which is around 462 pb! So I really do not get how they can merge their reads here...?? Or maybe there are pipelines to analyse non-overlaping reads but it seems a strange choice...

2) 515F and 806R (from https://earthmicrobiome.org/protocols-and-standards/16s/). They target the V4 region, with an amplicon size of ~292 pb,

which may cause trouble to merge the reads.

3) Finally, I was more tempted to use the 341F and 518R primers which target the V3 region (around 192 pb). But they are not degenerated.

Which primers do you use for this kind of application with the Iseq100?

Thanks a lot!