Hi,
With an in-house program developed by myself, the following information from the NGS result can be got:
ref_gi species genus %coverage reads_hit
Now the result suggests that a virus with coverage more than 90%. Then I assemble the reads guided by the reference, nearly complete genome of this virus has been achieved (Blast confirmed). My mates suggested me to perform a PCR.
In the table above, it showed 40%-60% coverage of several candidate viruses. I think here I need to perform a PCR to confirm it. Why I feel no necessary to confirm a virus with high coverage? The problem, perform a PCR to confirm bioinformatics analysis, sound like the statistical significance v.s. biological significance.
I could not answer the question. If we need to perform a PCR to confirm bioinformatics analysis every time, the NGS technology might simply provide a clue for downstream bio-experiment. With PCR result, it seems I can have more confidence. Can I have enough confidence on the analysis without PCR result?
Any helps from you will be highly appreciated.
Yang Li
Because of my job I know lots of clinical samples can not be characterized. A story happened to me is an infant suffered diarrhea with unknown reason. Colleagues here finally found out sapovirus infection after 7 days. During the 7 days, doctors used every methods to keep its life with a heavy cost (for the baby). I ask myself can we find out the reason more quickly?
NGS provided alternative method for virus identification and classification. The aim for the pipeline is, one command, taxonomy and genome coverage information can be achieved from a clinical metagenomic result. What are there in the samples are the basis for future analysis. However, currently no such a tool for clinical metagenomics data can be used to generate coverage information which I think it is quite an important parameters. I decided to develop one. After I finished it, I used sets of public data to test it and optimize the parameters based on results. Finally it works out.
Your words gave me a lesson. "The more skeptical you are of your own results early on, the easier it ends up being to convince others (including reviewers) later." I will figure out what to do. You have my thanks.
Very interesting, I certainly wish you luck with this, it has potential to be very helpful!
Thank you. I will release it on github when it is ready. I will appreciate your comments