16S primer pair

Hi all,

I would like to ask a basic question. Which primer pair do you guys use to do Illumina PE300 targetting 16S bacteria (specifically phytoplasma)?

Thank you

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Hello @amm59063,

We use the EMP Primers:

Lots of folks sell them, including IDT and zymo

They work well. And with Illumina paired end reads, you basically get 100% coverage of V4 in both directions, with makes pairing and error correction really easy. :+1:


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These are probably the most-used 16S primers out there for microbiome profiling, so make it easy to compare to other studies.

However, a word of caution:

The EMP primers also amplify chloroplast and mitochondria! Others may have specific recommendations, otherwise I recommend doing a literature search to choose either: (A) universal bacterial primers that do not amplify plastid/mitochondrial DNA or (B) blocking primers to eliminate amplification of non-target DNA. If you find any useful recommendations, please let us know!


Here is a nice table summary of some of the most common primer pairs you see around for target gene sequencing. As you’ll see there is no perfect primer pair and there is always some bias in the primers coverage. Now your job is to search the literature to see which primers work best (most coverage) for the sample type you are exploring. I do agree though the EMP V4 primers are the most common and is convenient for when you want to compare your study to other studies.


Thank you guys for the quick and resourceful answers. I’m going with 341F and 801R. I found a really good article(https://www.ncbi.nlm.nih.gov/pubmed/22933715) too.