Hello everyone, I have just started my PhD, and my project consists of analyzing fecal matter samples to characterize the intestinal virome in a population of individuals with metabolic syndrome.
I would like to request the guidance of some more experienced users because, throughout the steps for the analysis, I usually get lost as to what order the analysis should take.
I started with my FASTQ files and the next step I did was trimming my data, followed I using KneadData to remove traces of human DNA from my samples.
To evaluate the amount of human DNA I used bowtie2. Following this for the de novo assembly I used MEGAHIT and MetaSPADES to compare the information. I know that I should select just one.
After this step, I started to have problems regarding which route to take and what software to use, I have relied on several manuscripts but usually end up more confused.
I have read these two papers and some more but the more I read the more doubts I have:
de Jonge, P. A., Wortelboer, K., Scheithauer, T. P., van den Born, B. J. H., Zwinderman, A. H., Nobrega, F. L., ... & Herrema, H. (2022). Gut virome profiling identifies a widespread bacteriophage family associated with metabolic syndrome. Nature Communications, 13(1), 3594.
Johansen, J., Atarashi, K., Arai, Y., Hirose, N., Sørensen, S. J., Vatanen, T., ... & Plichta, D. R. (2023). Centenarians have a diverse gut virome with the potential to modulate metabolism and promote healthy lifespan. Nature Microbiology, 1-15.
For example, once you have the de novo sequences, some recommend using circlator to detect circular sequences, I don't know why. It is not clear to me why identify circular sequences.
For example, some do not do this step but after having the de novo sequences they use VirSorter v1.0.6 saying that at the same time they use Virfinder, my question is if I first run my information in Virsorter and then in Virfinder and later with the software CAT, BAT, and RAT
In general, after having the de novo sequences, I began to have problems regarding how to carry out my analysis and which route to take. I have relied on some papers but I have ended up with more doubts and questions than answers, any suggestions or guides in this regard?
I would be very grateful
Best regards.
