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:
- What do I actually need to order?
- What are some good companies to order from?
- Is qPCR necessary for this type of analysis ?
