I wanted to do the virome analysis including DNA/RNA bacteriophages and other viruses, microbiome, yeast. What is best approach? Whether we can do it in single reaction or separately, without compromizing the quality of data.
Hello, nowadays net generation sequencing techniques fully applicable for microbiome analysis including yeasts, bacteria, bacteriophages, in single reaction. Ion Torrent sequencing, Ion Semiconductor Sequencing or Pyrosequencing.
It sounds like you want to sequence bacteria and viruses from the same sample, right? You can of course do a metagenome in one go, but you will miss some of the relevant taxa for viruses (for example RNA viruses). Also some taxa will be higher represented in the data than others due to their abundance, so you will miss the lower abundant viruses that will get less read coverage.
It depends a bit on the sample and question that you are asking. Generally I would say, that if you can separate your sample into a bacteria fraction and virus fraction, it will give you best data.
For example, some use ultracentrifugation with CsCl gradients. You would take the fraction with your intact virus capsids and do DNA and RNA extractions from it. Sequence these along with your DNA extraction of the bacteria fraction. Depending on the extraction protocol, you might introduce biases towards ssDNA, dsDNA, ssRNA and dsRNA viruses. So you probably want to have a good look into it.
Check out this paper here as a start:
https://www.frontiersin.org/articles/10.3389/fmicb.2014.00206/full
Go with NGS separately for best data, while you can go with mixed too but data reliability will be questionable. further, 2-5% of the sample will be verified using Sanger before making any conclusions
Best of Luck.
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