Can MiSeq Reagent Kit v2 (500 cycles) be used to sequence V3-V4 region of bacterial 16S rRNA gene?

General Discussion
1

Hi everyone!
I am about to carry out my first amplicon sequencing run. Can someone please guide me if I can sequence V3-V4 region of the bacterial 16S rRNA gene using a MiSeq Reagent Kit v2 (500 cycles i.e. 2x250bp)?

The average size of the V3-V4 amplicon according to the Illumina guide is approximately 460bp.

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Hi @ahsankaleem,
While in theory this sounds like it would work fine since 500-460=40bp to spare across both reads for merging/trimming etc, in practice this is a bad idea because the tail 3' end MiSeq runs often drop significantly in quality to the point where you really need to trim them. Failure to trim those poor quality tails leads to you losing many/most of your reads during QC, and those that pass will also be biased against clades that may be naturally longer than 460bp in their V3-V4. I would strongly suggest running them on a 2x300 runs, or, just use V4 if you want to avoid the extra $.

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Thank you very much for sharing your valuable opinion. What do you think about using V1-V2 or V2-V3 region instead of V3-V4 region?

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Hi @ahsankaleem,
It really depends on your target community, and what you plan on doing with the data. If the objective is to compare the results to other data, then the EMP V4 (and its extension V4-V5 if 2x300 bp sequencing) is by far the most popular ones and thus has the most public data out there to compare to. But, there may be cases or communities where other primers perform better, but you'll have to dig into the literature yourself and see the best choice for your study.

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Thank you very much for your guidance. It will help a lot.

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