Selecting new primers for Illumina's 16s protocol

[giderk]

This is a quick question with hopefully a quick answer

Illumina published a recommended protocol for 16S Metagenomic Sequencing Library Preparation. In the protocol they suggest that you can swap in your own primers, just add on the appropriate overhang sequences.

Their bulletin on this protocol says..."Locus-specific portion of the forward and reverse primers (NOT including the overhang sequence) must have a melting temperature (Tm) of 60°C to 65°C. If melting temperature is lower than 60°C, Tm can be increased by adding several “padding” nucleotides."

Why would this be? Does this mean that the overhang adapters interfere with primers that amplify at conditions other than what is recommended?

I'm interested in using this protocol with the primers used by Mahe et al 2015, Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐rDNA in Environmental Microbial Eukaryotic Diversity Surveys

[JBKri]

I too wonder about this! I think it might be a mistake. It would make sense if the sequencing primers were the same as the locus-specific portion (as in Kozich design). If not using a custom sequencing primer it should not affect the sequencing. And the reverse primer in the above Illumina document doesn't have a melting temperature in the suggested range (it's 51.3 C according to IDT page), so they apparently don't follow their own recommendation!

We used these exact primers and the Illumina protocol, and we got excellent results. So I don't think the melting temperature is critical.

[JBKri]

I got this response from Illumina Tech Support regarding the melting temperature:

"The reverse primer in the 16S protocol is a well known standard primer (legacy
primer) that was used in the field for many years and, while it does not fall
within the specs we normally recommend, we did much testing on the primer and
found it to work well.
Other primers outside the 60-65 C range may work as well, but we are just less
confident in primers with more extreme Tms.

However, in general the best practice for customers is to test and optimize
the primers they are planning to use on their specific samples, as different
organisms mixtures might present different features in terms of sequence
specificity and an initial optimization will maximize the probability of
obtaining as much information as possible from their experiments."

So, not very clear, but it seems their concern is about primer specificity in mixed template samples.

[thermophile]

the tm of your sequencing primer is really critical if you're going off the Kozich design. My first custom amplicons with that design, I didn't check the sequencing tm just the overall and the sequencing didn't work (sequencing primer tm was ~70)