Question: Metagenomics Analysis
3
gravatar for Bdv
9.0 years ago by
Bdv310
Bdv310 wrote:

I want to sequence in 454 for microbal diversity. Is it better to sequence 16srRNA or I can sequence everything and then use a software like CARMA which uses protein encoding reads to make the taxonomy analysis. What is the preffered way? if the 16s rRNA , which software is recommended for the analysis?

rrna metagenomics taxonomy • 2.9k views
ADD COMMENTlink modified 8.8 years ago by Pawel Szczesny3.2k • written 9.0 years ago by Bdv310
3
gravatar for Prohan
9.0 years ago by
Prohan350
United States
Prohan350 wrote:

If you just care about diversity and not too much about function - I would go with 16S. Pick a good primer set though. You can check how well it covers known 16S by using the Seqmatch tool at the Ribosomal Database Project.

It's also really important to define what an OTU - probably best to just use the ICOMM primer set as they've done a lot of work on determining what percent identity to use for the V6 region. With that said though their primers were designed for first gen 454 and only give something like a 50bp product.

Mothur is a good place to start for high throughput 16s analysis.

ADD COMMENTlink written 9.0 years ago by Prohan350
1
gravatar for Pawel Szczesny
9.0 years ago by
Pawel Szczesny3.2k
Poland
Pawel Szczesny3.2k wrote:

I would say that you need to choose between coverage and accuracy if you are deciding between these two approaches. Sequencing of 16S RNA gives you high degree of accuracy (if you use high quality reference database such as Silva), although coverage might be an issue (we get constantly higher diversity from generating several set of primers targeted at different parts of taxonomic tree than from using universal 16S RNA primers). When sequencing everything, you skip filtering-by-amplification step, so in theory you should have higher coverage (I'm not sure it has been tested, but that would be my intuition) although I would question accuracy, as protein databases are not that strict about taxonomic assignment (I would be skeptical about taxonomic origin of a protein sequence coming from an organism that doesn't have complete genome sequenced and assembled).

If you don't expect a large number of weird things in your sample, go for sequencing 16S RNA. For that Mothur is indeed a very nice tool and make sure you visit Mothur's wiki especially Example data analysis page.

ADD COMMENTlink written 9.0 years ago by Pawel Szczesny3.2k

I disagree about the assumption that you are going to get more coverage if you sequence everything. By sequencing the whole genomes of multiple bacteries, you will probably end up with very low coverage throughout the genome. With 16S only, you are going to get much higher coverage for the sequence of interest. Coverage is defined here and here.

ADD REPLYlink modified 3 months ago by RamRS25k • written 8.8 years ago by Eric Normandeau10k

Eric, I was referring to taxonomic coverage not sequence coverage. You get consistently more OTUs from WGS than from 16S sequencing.

ADD REPLYlink written 8.8 years ago by Pawel Szczesny3.2k
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