Fungi primers for MiSeq

Question

[seelan80]

HI All,

As a beginner to MiSeq, I would like to know how we can choose primer amplicons for MiSeq for environmental samples such as waste water to analyse fungal communities. Your help is highly appreciated.

Answer 1

[agata88]

You can identify your fungi by ITS analysis, in that case you should look for primers that have been already projected (in some publications, e.g. "Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts") to for example S288C strain (Saccharomyces cerevisae). It is the most common technique to find the composition of fungal population in samples. The ITS fragments are not very long so It will be pretty easy to run it on MiSeq.

Hope it helps,

Best,

Agata

Answer 2

[fanli.gcb]

We've used the BITS primers discussed here: http://aem.asm.org/content/79/8/2519.full

Note that ITS amplicons vary quite a bit more in size than your typical 16S amplicons, so you should be aware of this when designing your sequencing experiment. 2x150bp often doesn't span the full amplicon so you have issues with read merging. 2x250bp v2 is probably your best bet at this point, as the 2x300 kits are still problematic.