16S metagenomics on MiSeq

[sajoshi]

I will be doing MiSeq run for 16S metagenomics. it will be my first time. Recently some MiSeq v3 300 PE runs gave me trouble and hence I am worried. Are there any things I should do differently from what the 16S protocol says. Illumina asks to spike at 25%, should it be that high.

What other things should be taken into consideration while preparing the libraries. I will have at least 96 samples. Can I pool all on PE 300 v3. Please advise and provide feedback/suggestions, anything and everything there can be for this kind of run.

Thanks in advance

[fanli]

There are a ton of threads discussing issues with the 2x300 v3 kits:

http://seqanswers.com/forums/showthread.php?t=59558

http://seqanswers.com/forums/showthread.php?t=67336

[kmcarr]

Recently some MiSeq v3 300 PE runs gave me trouble and hence I am worried. Are there any things I should do differently from what the 16S protocol says.

Yes, there are. For starters, absolutely do not use the v3 300PE kits for 16S amplicons. Stick to the v2 PE250 runs.

Illumina asks to spike at 25%, should it be that high.

5-10% PhiX spike-in is sufficient.

[thermophile]

I've had to go to 20% phix for 16S runs otherwise R2 is horrible.