Illumina adapters with custom PCR primers

[hmoranmac]

Hi all,

I am in the amplicon PCR stage of amplifying fungal DNA. However, I need to order primers with Illumina adapters on them, as I am planning on sequencing down the line. I am following a fungal metagenomics library prep workflow for referring to the Illumina adapter sequences. The protocol says I can use a custom locus-specific primer; "The overhang adapter sequence must be added to the locus-specific primer for the region to be targeted".

However, when I am adding the Illumina adapters to my sequence in IDT (ordering 25 nmol), the reverse is 61 bp long and gives me a warning. Can someone help me understand how to order the primer with adapter attached, and if I can check their compatibility?

Thanks xx

[Tcom111]

Hi,
I guess a good solution for this is a two-step PCR. The first is with custom locus-specific primers with the 5' addition of a partial Illumina sequence. The other set is a full Illumina adapter sequence that contains an index as well. That way you can use the same pair for all of your samples in the 1st PCR, and index your reaction in the 2nd.
Some more explanations and example sequences can be found here:

https://pubmed.ncbi.nlm.nih.gov/27558250/

good luck

[Seqing_guidance]

Hi there,
Are you amplifying/sequencing fungal ITS or another gene? Just curious. Are you using the pooled primers recommended in the protocol? The first step PCR uses your regular fwd and rev primers (or the pooled primers) and the HotStart ready mix ads the necessary (short) sequence that the Illumina indices will then attach to in the 2nd PCR reaction.