[DIYbio] Re: 16s + ITS Primer Ordering Help

Here are the 16s primers(newest versions from literature I think) if you want to order them from of or just order them yourself
https://www.the-odin.com/bacterial-16s-rdna-primers-for-bacterial-identification/

ITS Primers(I think we use older ones and I can't remember if they have changed recently)

https://www.the-odin.com/fungal-its-pcr-primers-for-identification-and-barcoding/

Is qPCR nec.? Depends on your goals or what information you need.

If you are new to microbiome analysis, I really recommend using Argonne National labs. Especially if you are going for any sort of quantification because there are so many things you might miss and it only costs $6 / sample for library prep for 16s or ITS.

http://ngs.igsb.anl.gov/

Josiah Zayner, Ph.D.

The ODIN, Founder & CEO

http://the-odin.com

On Tuesday, January 3, 2017 at 1:09:53 PM UTC-8, Emerick Larkin wrote:

Hello, I am a graduate student starting a research project involving the characterization of plant microbiomes. I have several genomic DNA samples, isolated using the MOBIO power soil kit and wish to move on to a general analysis of the microbial community composition, from a fungal and microbial perspective.

I plan on doing this initially with separate 16s and ITS variable region amplification and subsequent analysis using the SILVA and UNITE databases, respectively.
 
I plan on using my universities MiSeq for the sequencing, once I have around 100 samples. However, I would like to try some preliminary PCR on my early samples, to verify that the samples aren't full of inhibitors.

I have been looking at the Earth Microbiome project and would like to follow their protocols for library prep and miseq sequencing. They indicate that they use the 515f-806r primer pair for 16s and ITS1f-ITS2 pair for ITS. They have spread sheets for these primers at the follow links:

http://www.earthmicrobiome.org/emp-standard-protocols/16s/
http://www.earthmicrobiome.org/emp-standard-protocols/its/

There are many sequences listed in the 16s excel spread sheet and for 16s  there are both 'amplification primers' and 'sequencing primers' listed on the webpage. There are also several different sequence  primers listed on the ITS page.

My questions are:

1. What do I actually need to order?
2. What are some good companies to order from?
3. Is qPCR necessary for this type of analysis ?