Illumina MiSeq 16S sequencing EMP protocol questions

SDPA_Pet · 04-14-2019, 04:07 PM

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 5–10% 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, 09:03 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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04-14-2019, 04:07 PM	#1
SDPA_Pet Senior Member Location: US Join Date: Apr 2013 Posts: 222	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 5–10% 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?

04-14-2019, 09:03 PM	#2
ikripp Member Location: QLD, Aus Join Date: Jan 2018 Posts: 19	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