Questions about V3–V4 primers for 16S rRNA amplicon sequencing, and calculating overlap

[benjjneb (Ben Callahan)]

Yes to basically all of that.

One addition of actual substance:

@KQUB

The Klindworth et al. paper gives an amplicon size of 464 bp for these primers, whereas the Thijs et al. paper gives an amplicon size of 444 bp. If the primers, plus the sequence they amplify, span from positions 341 to 805, I guess the correct amplicon size should be 805 − 341 = 464 bp, right? Also, what does this number mean exactly? Is it the amplicon size expected if the primers are used to amplify from the E. coli 16S rRNA gene? And, if using these primers to sequence an environmental sample, would we then get a range of slightly different amplicon sizes (because the 16S rRNA gene varies slightly in different species)? In other words, is 464 bp kind of an approximate number?

Yes, there is variation in lengths of 16S segments. It isn't large, but it exists. In particular, there are two modes of V3V4 length in nature, one at 460 nts and another ~440 nts (using these primers). So you'll want to make sure even the longer natural amplicons will sufficiently overlap after truncation.