Metagenomics with Illumina, all species genomes coverd by reads or not?

Question

[m.koohi.m]

Hi All,

I am new in Metagenomics and want to ask a basic questions!

suppose we have a microbial community and use Illumina for WGS. We don't know any things about our community and don't know which species are there (Actually it is a de novo sequencing). I want to know is there any possibility that there are some parts of our unknown bacteria genome that is not covered by reads produced by Illumina? Or for sure all part of species genomes will be covered by reads?

P.S: I read some articles about the library preparation in Illumina WGS. I were wondering if there is any relation between this issue and library preparation?

Thank you,

Answer 1

[Steven Lakin]

As Daniel mentioned, the deeper you sequence, the more representative of a population you will have in your data regardless of library preparation, especially with WGS metagenomics. There is a measure called rarefaction that tracks this in ecology (which is essentially what you're doing with metagenomics). Take a look at a few papers on metagenomics measures. A few other points as well: you probably wont be able to get coverage for de novo assembly (whole genome mapping) and wont be able to entirely separate out one organism vs another. Many metagenomics approaches rely on binning instead. There are some comprehensive reviews out there that you should take a look at before designing your experiment. A search for metagenomics review or primer will pull them up.

[m.koohi.m]

Thank you for your answer, Thank you for keywords that you mentioned to further study.

Answer 2

[User 59]

Yes it is possible that you will miss parts - this is because you're sampling by sequencing. In order to be sure you covered everything you would potentially have to sequence very deeply. Some organisms will be present at low levels, so how much sequencing would you need to do to a) detect them and then b) ensure they were fully covered? This would be a case of diminishing returns.

Whether this is an issue of library prep or not is almost immaterial.

[m.koohi.m]

Thank you for your answer. Got it! Just one question, what is the exact meaning of "Some organisms will be present at low levels". What is the main reason that some organism can detect easily but some of them not? It depends on their abundance in that community or any other things?

[User 59]

Yes it depends on their community abundance, sorry if that wasn't clear from my reply.