Dear Charlotte

I think that the topic of gut microbiota functionality is very interesting. I think that with NGS you could get a lot of information about the function of the organisms in your samples. You have at least two options:

1) You could perform a whole metagenome sequencing (WMS) by sequencing the DNA of all the species in your sample. In this case you could identify most of the present species, getting up to the strain depending on the depth of sequencing and the bioinformatics pipeline. In addition you could get an indirect functional analysis because you could identify the genes associated to the different taxa and then infer their function.

2) The most direct way to address the functionality problem would be through a metatranscriptomics analysis. In this case you would sequence all the transcripts expressed by the microbiota. Thanks to this approach you could not only identify the species in your samples (even though their quantification might be tricky) but also quantify the expression levels of the genes they express. Then by associating genes to functions you could "easily" correlate taxonomy with functions.

We have analyzed several metagenomics and metatranscriptomics data and I can tell you that you can obtain a nice picture about the function of the species and the pathways activated in your samples. With a proper experimental design you could also perform a differential analysis to identify functions which are associated to a specific group of samples.

We have recently developed an online and user-friendly tool to analyse amplicon, WMS and metatranscriptome data, if you want you could give a look at it: https://metagenomics.sequentiabiotech.com/

I hope this might be useful.

Have a nice day

Riccardo

Bioinformatics data analyst at www.sequentiabiotech.com

NGS is better than DNA microarray for your work.

Thanks a LOT guys !!

Riccardo, thank you very much for these two options. What are the pro(s) and con(s) of each method (metatranscriptomics vs. WMS) ? For instance, which one is the most expensive (I have a lot of samples :O)?

Would you have an article that would compare these methods?

Again thank you very much !

Charlotte

Dear Charlotte

you are welcome :)

In my opinion the pro(s) of metatranscriptomics is that you can really focus on the genes which are expressed in the community, so you have a direct indication of which metabolic pathways are active and which are silenced. In addition you can understand how microbes "complement" each other by producing metabolites that can than be used by other species in the community. The drawback is that it is more difficult to quantify the abundance of the species and in the end it might be difficult to detect lowly expressed genes (unless you sequence a lot of reads). The final drawback is that it is at least 25% more expensive than WMS.

On the other hand WMS has the advantage that let you identify and quantify most of the species. The functionality however is indirect, so you infer that a species is having an activity because it contains the genes to do it.

Here you can read more:

Article Use of Metatranscriptomics in Microbiome Research

https://www.nature.com/articles/ismej2014245

https://www.nature.com/articles/nrgastro.2018.15

In case you need more help do not hesitate to write me privately.

Have a nice day

Riccardo

Bioinformatics data analyst at www.sequentiabiotech.com