Tm for PCR1 primer for amplicon-seq

[Kritee M]

Hi all
Total Next-seq newbie here! The Illumina protocol for targeted amplicon seq says that - The locus‐specific portion of primer (not including overhang sequence) must have a melting temperature (Tm) of 60°–65°C.
With a standard 17-21 bp primer length, my Tm comes to be around 53-55. I have to really increase primer length till 36bp (which makes no sense) to get a Tm of around 60-62. Does anyone have experience using lower than recommended Tm primers for amplicon-seq. How should I navigate from here ? Will appreciate any suggestion. Thanks!

[ADoetsch]

We're using the protocol by the Schloss Lab since many years now and had good results with adapting the protocol to various target regions (e.g., 16S V4, ITS1&2, trnL). There exists a very well written SOP for using the protocol and designing your own primers. It's written for the MiSeq platform but most details should directly translate to NextSeq. You can find it here: https://github.com/SchlossLab/MiSeq_..._WetLab_SOP.md

The melting temparture of 65 °C usually refers to the binding temperature of the sequencing primers because there is no way to change that in the Illumina platform itself. In the Schloss protocol the sequencing primers consist of the locus-specific sequences, a 2-bp linker and a socalled "pad" region whose purpose is to get the melting temperature right. I'm not sure about the procotol you mention but I assume that it's also about the sequencing primers.
In principle, you could also design the locus-binding regions longer to achieve the melting temperature but I suppose that it would decrease primer sensitivity since longer sequences increase the amount of species that don't fit your primer sequence and won't be amplified.

The purpose of the 2bp-linker between pad and target region is to prevent amplification bias. The two basepairs are chosen so that as few as possible of the targeted species match their sequence, so that the length of the binding isn't increased by a chance sequence match upstream of your binding region.

Long story short: include a pad region in your amplicon primers (and sequencing primers) and adapt its sequence to get the right melting temperature.