Hi, Can NGS analysis with 16S rRNA gene sequencing get microbiome information of both Bacteria and Fungus with same primer set?? I mean when you do microbiome analysis(soil or feces), how do you get the comprehensive microbiome information with NGS analysis, because there exist both Bacteria and Fungus.
Could you please provide us with some more information? I am not sure I understood your problem.
Did you do a 16S rRNA gene amplification and sequenced the amplicons? If yes, rRNA sequences from both, Bacteria and Fungus, might be a result of a primer bias? Meaning it might be possible that your primer pair also amplified some 18S rRNA sequences.
May I ask which primer pair you used to generate the 16S rRNA gene sequences?
I would recommed to evaluate your primer pair of interest using SILVA TestPrime
http://www.arb-silva.de/search/testprime/
However, please be aware that an in silico evaluation doesn’t guarantee good lab results. The results are based on a computer model and cannot represent the real world. Nevertheless It helps you to choosed the most suitable candidates out of a large number of possibilities and also check if your choosen primer pair might fail to detect the taxa of interest.
Thanks for your answers.
My primer set is below:
CS1-341F: 5'-ACACTGACGACATGGTTCTACACCTACGGGAGGCAGCAG
CS2-806R: 5’-TACGGTAGCAGAGACTTGGTCTGGACTACHVGGGTWTCTAAT
I mean, is this primer set suitable for Fungus, and i will do the NGS sequencing on Ion Torrent platform, Can i get the microbiome information on both Bacteria and Fungus? Thanks!
I don't think so. Frequently, the bacterial primers get mitochondria and specially chloroplast DNA amplification but aside from that...
And you still would need the archaea primers. In feces they are important, and depending on the soil they can be important too.
I just found this paper that you may want to look at. PLOS one: Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing
Article Development of a Prokaryotic Universal Primer for Simultaneo...
16S is just for Bacteria and Archaea. There is no sense on this... If you wanna look to Bacteria and Fungi at the same time you could perform shotgun metagenomics with random primers and barcoding, via high-throughput sequencing (e.g. Illumina). But, you need to keep in mind that there is no database where you can input your sequences and have good results of taxonomy for both Bacteria and Fungi. Most of people use MG-RAST as server and match the sequences to the best hit M5nr non-redundant database (best hit from 5 databases) or single databases (SEED, KOGG, etc). Then, you will have a good match for Bacteria but not that good for both Archaea and Fungi
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