I'm trying to perform a survey of the life in a soil sample. I want to know what species of bacteria, fungi, and other organisms are in it. I've heard that ITS sequencing and 16S rRNA sequencing are both possible ways of doing this, but I don't know enough about these techniques to understand which (if either) I should use, or how you interpret the resulting sequence file delivered by the lab. Will these techniques do what I want? How do I choose one over the other? And how do I interpret the results?
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2 Answers
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16S and ITS techniques try to identify organisms in your samples by amplifying short, 'barcode' sequences from each organism's DNA, and using those short sequences to try to identify the organism they came from.
16S can be useful for distinguishing between prokaryotes (bacteria), at least to genus level. It will not always give enough information to determine exact species though. ITS can be particularly useful for distinguishing between some eukaryotes (plants, fungi, etc), even down to species level. If you are interested in both bacteria and fungi you would need to carry out both analyses on your samples.
A different approach is to use shotgun metagenomics, where rather than amplify only the 16S or ITS region, you extract and sequence any DNA you can from the sample, and then try to assign each sequence read to a species/genus by alignment with a reference collection (e.g. MEGAN pipeline) or by kmer analysis (e.g. Kraken).
There are some packaged approaches for dealing with 16S. Qiime2 is popular, and will likely involve some amount of work in python to get it working. There is an R package microbiome, but I don't have experience with this. Mothur is a long-standing stand-alone package that is resonably easy to use, and MG-RAST is a web-based version that is also reasonably easy.
ITS analysis is less fully developed as pipelines, but I am sure you can find some in the literature by searching for other people's studies of soil microbiomes.
This review of techniques and approaches might be helpful if you are interested to learn more about the whole genome/metagenomics approaches: http://ccb.jhu.edu/people/salzberg/docs/Breitwieser-etal-2017-Metagenomics-review-reprint.pdf
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16S is still one of the most poplular methods of idenifying organisms. The technique is very well stablish and it works well. However, it has some limitations, for example there is a significant variance of the copy number of these gene in the species. Which makes quantification not very reliable.
To decide which tools are better to analyise your results, you can have a look a the CAMI challenge paper. Several tools for different parts of the analysis are compared across labs. The most popular tools are not always the best for your analysis.
