Hey there,
I want to analysis a soil's microbiome and so far used 16S and ITS primers on DNA extracts (later RNA as well) to tackle bacteria, archaea and fungi.
Due to problems with the size of the complete ITS region for sequencing, I was looking into alternatives for the eukaryotic partition of the samples. So far that includes just focusing on primers for ITS 1 or 2, but also 18S. As 18S also has the advantage of including micro-algae and protozoa, I wondered:
1) Why it isn't used that much. Are there problems in the methodological approach or trustworthy compatibility from 18S data?
2) Why is ITS believed to be more reliable for metagenomic amplicon approaches? I couldn't really find this, as 18S looks like giving a way more complete picture. Please feel free to share your experiences (and I am heading for Illumina MiSeq).
Thanks a lot :-)
For fungi, ITS has a good reference database ( https://unite.ut.ee/ ) and has been widely adopted as a the best marker gene ( Article Nuclear ribosomal internal transcribed spacer (ITS) region a...
).As far as usage of 18S I think it is just a matter of more people doing human/animal model related microbiome research. I have seen 18S used in a number of aquatic microbiome studies. I am not as familiar with the soil literature.
¨Dear Christian,
thanks for your input. Yes, I did not consider the database bias in using 18S when it comes to mammalian metagenomics etc. I heard about this unite database, but good to see it indeed is in use.
I will still try to find a way with 18S primers on v3 Illumina MiSeq I guess ;-)
Best,
Maria
Maria Scheel
Although Christian Edwardson gave you the answer, still I want to add few things
- I suppose what is metagenomics and you are aware of the term you used "mammalian metagenomics" is not really valid.
- you can look on the very good explanation about similar query in the discussion https://www.researchgate.net/post/what_is_the_difference_of_16s_rRNA_and_18s_rRNA_Which_one_is_suitable_for_bacteria_associated_to_coral
- As Christian said, it is not really a "trust worthy" thing, it is actually the thing we want to cover in the sequencing. Theoretically "Universal" (so called) 16S primers should cover all the bacterial/archaeal diversity, however, it is seen that universal primers do not cover them equally well. That is why there are archaea specific 16S primers (this also do not cover well some specific archaeal groups). Therefore, there is another marker "mcrA" used frequently for archaeal studies.
- Similarly, with more and more fungal studies it is experienced that ITS primers covers the fungal diversity better than other markers. Hence it is not the question about reliability but the coverage of diversity. One more thing here is the sample. If you have something not very common, the coverage might vary. Here I mean to say that, choice of marker depends on the diversity you want to cover.
- You actually can't use one marker for all the target organism group, there are a lot of factors to consider. So, make a choice based on those parameters.
Maybe the title of my question is not so much clarified by my text, but I am looking into alternatives for microeukaryotes and hence the different use of the 18S and ITS primers; not 16S and 18S, which are tackling different areas.
Thanks for clarifying, Abhijeet Singh but while my word usage might not be correct, I simply wanted to hint at the bias towards mammals in 18S databases.
So, I basically was interested in finding a primer to tackle microeukaryotes without ignoring the non-fungi geni - which might have been 18S, but in literature got the notion, that it is not the preferred primer. So, I am just interested finding a way to tackle more eukaryotes than just funig basically.
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