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Old 04-04-2016, 11:51 AM   #1
sajoshi
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Default 16S metagenomics on MiSeq

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
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Old 04-04-2016, 04:13 PM   #2
fanli
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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
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Old 04-05-2016, 07:50 AM   #3
kmcarr
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Quote:
Originally Posted by sajoshi View Post
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.

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Originally Posted by sajoshi View Post
Illumina asks to spike at 25%, should it be that high.
5-10% PhiX spike-in is sufficient.
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Old 04-05-2016, 09:43 AM   #4
thermophile
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I've had to go to 20% phix for 16S runs otherwise R2 is horrible.
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