First, how to isolate DNA from effective microorganisms? is it the more to genomic DNA isolation or metagenomic DNA isolation protocol?
Second, is the Bacterial 16s rRNA Amplicon Sequencing with Illumina 250 PE/ 300 PE strategy, suitable to study their microbial diversity?
In my view, a combined SDS- CTAB method could serve the purpose of DNA extraction from the mixed culture of EM. The following link has some detailed procedures which could be of your help.
https://www.ijcmas.com/6-3-2017/Swapan%20K.%20Tripathy,%20et%20al.pdf
For your second question, 16S rRNA gene Amplicon sequencing would not be an appropriate way to study the microbial diversity of EM since they also have fungi and Yeasts. So you can opt for whole metagenome sequencing of EM.
Goodluck Farah Fadwa Ben belgacem
1. You can use either CTAB method or phenol-chloroform method for isolation of DNA from individual bacterial strains and different kits, such as FastPrep DNA, DNeasy Power etc. are available commercially for extraction of metagenomic DNA from environmental samples.
2. To study microbial diversity, 16S rRNA based Illumina sequencing 300 PE is better option as compared to llumina sequencing 2500 PE
Rachel Samson.P
Your assertion and reason opposing 16S rRNA gene sequencing does not make sense.
Abhijeet Singh
To my knowledge, Effective Microorganisms (EM) are mixed cultures of beneficial naturally-occurring organisms that can be applied as inoculants to increase the microbial diversity of the soil ecosystem. They consist mainly of the photosynthesizing bacteria, lactic acid bacteria, yeasts, actinomycetes, and fermenting fungi. So if only 16S rRNA gene sequencing is done won't the fungal and yeasts get excluded, which are usually identified by amplifying ITS regions? Therefore I recommended WGS.
Rachel Samson.P
Yes, now it is clear what you wanted to say.
Since question itself is not clear what diversity needs to be analyzed. But since, the questioner mention for only bacteria, it can be interpreted as she is only interested in bacteria. In this case, 16S rRNA only is enough.
Rachel Samson.P
Thank you for your clear answer. It is true what you said about the presence of others than bacteria in EMs and your remark was made correctly, I am interested in bacteria. Currently, I am not able to do WGS due to the high cost, therefore I want to make sure if the Bacterial 16s rRNA Amplicon Sequencing is good enough to study the microbial diversity.
Very cordially.
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