16S rRNA micobiome project: Which primers to use?

Question

[Grant]

Hi guys

I'm completely new to the field, and am hoping to start a project soon examining the oral, airway, and gut microbiome of TB patients.

I'm seeking advice on which primers (targeting specific regions of the 16S RNA gene - V1, V2, V4 etc.) to use. There doesn't seem to be a good consensus in the literature.

I am aware that different primers offer different levels of discriminatory power and, if the amplicon is too long, it is difficult to get good quality sequence from a single read near the end of the strand (and discriminatory power is subsequently lost). Is this accurate?

We plan on using an Illumina HighSeq instrument, but also have a MiSeq available. Which machine would be better for this purpose?

Thank you!

Answer 1

[marina.v.yurieva]

We usually use V4-V5 primers for 16S (oral and gut microbiome). People sequence different regions for different microbiomes, so for gut it is usually V4-V5. MiSeq is good enough but you can use HiSeq if money is not an issue. For me it seems like an overkill but some people do use Hiseq. Plus, Miseq reads are longer. I find it better to use as long reads as possible (be careful with Illumina though: there is some issue with 300bp reads sequencing going on now and they are still working on fixing it). So I usually use single 250bp reads.

Good luck!