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-   -   Illumina MiSeq 16S sequencing EMP protocol questions (http://seqanswers.com/forums/showthread.php?t=88788)

SDPA_Pet 04-14-2019 03:07 PM

Illumina MiSeq 16S sequencing EMP protocol questions
 
Hi, I'm going to do some 16S barcoded PCR following the EMP standard protocol here (http://www.earthmicrobiome.org/proto...standards/16s/).

The PCR potocol seems straight-forward. However, I am confused about what they say this on the website -- " For running these libraries on the MiSeq and HiSeq, please make sure you read the supplementary methods of Caporaso et al. (2012). You will need to make your sample more complex by adding 510% PhiX to your run."

I have a few questions:

1>I google the Caporaso paper and it seems PhiX is some kind of control?

2> At which step, should I add this PhiX? Do I have to add it or it is optional? Do I need to add this into my PCR master mix? -- add it in to every samples?

3>5-10% PhiX is the final concentration of PhiX or the volume of concentration the original PhiX?

4> Where can I order the PhiX or Is this a kit?

ikripp 04-14-2019 08:03 PM

PhiX is the control spike in from Illumina, which you can order from them. It is recommended for use with low diversity libraries and is added following the library denature and dilution. More info can be found here: http://sapac.support.illumina.com/co...5039740-10.pdf


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