What are the best barcoding - multiplexing strategies?

Hi Qiime2 forum folks,

I considering launching a new project including 16s amplicon studies, using hundreds of samples from rhizosphere soils.

If you had to start from scratch, which barcoding - multiplexing strategies would you consider best?

With a limited budget, which primer sets get you the most samples for your money?

Thanks in advance for your help,

best wishes



Hi @mshenton,

The EMP protocols are very commonly used for 16S V4:

Commonly used translates to fewer bumps down the road in figuring out protocols and analysis — so I’d recommend this as a tried and true method.

That said, some other schemes like dual-indexing can help decrease sequencing error slightly, but these come at greater expense since all primers are barcoded. I am hesitant to endorse any particular method without having tried them all.

There is a wide range of different methods used out there and surely an equally wide range of opinions — let’s see if others on this forum have any opinions (and especially literature benchmarking different methods).


Hi @mshenton,

The following Nature Biotechnology article compares the EMP 16S Illumina Amplicon Protocol with a dual-indexing approach outlined in an Illumina technical note. It also investigates various factors such as the number of PCR cycles and type of polymerase used (Taq vs. High-Fidelity) and the impact on errors and biases using mock communities.

Gohl, et al (2016) Systematic improvement of amplicon marker gene methods for increased accuracy in microbiome studies, Nature Biotechnology, 34(9), 942-952


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