Since metatranscriptomics focuses on what genes are expressed, it enables the characterization of the active functional profile of the entire microbial community.
[4] The overview of the gene expression in a given sample is obtained by capturing the total mRNA of the microbiome and performing whole-metatranscriptomics shotgun sequencing.
[6] A typical metatranscriptome analysis pipeline: The first strategy maps reads to reference genomes in databases, to collect information that is useful to deduce the relative expression of the single genes.
One of the latest metatranscriptomics techniques is stable isotope probing (SIP), which has been used to retrieve specific targeted transcriptomes of aerobic microbes in lake sediment.
The Trinity software for RNA-seq, in comparison with other de novo transcriptome assemblers, was reported to recover more full-length transcripts over a broad range of expression levels, with a sensitivity similar to methods that rely on genome alignments.
[18] HUMAnN2 is a bioinformatic pipeline designed from the previous HUMAnN software, which was developed during the Human Microbiome Project (HMP), implementing a “tiered search” approach.
This pipeline is highly flexible, since it offers the possibility to use third-party tools and improve single modules as long as the general structure is preserved.
[12] The mOTUs2 profiler,[22] which is based on essential housekeeping genes, is demonstrably well-suited for quantification of basal transcriptional activity of microbial community members.
However, microarrays are affected by some pitfalls: RNA-Seq can overcome these limitations: it does not require any previous knowledge about the genomes that have to be analysed and it provides high throughput validation of genes prediction, structure, expression.
Its prevalent functions are related to the fermentation of indigestible food components, competitions with pathogen, strengthening of the intestinal barrier, stimulation and regulation of the immune system.
[23][24][25][26][27][28][29] Although much has been learnt about the microbiome community in the last years, the wide diversity of microorganisms and molecules in the gut requires new tools to enable new discoveries.
This suggests that non-housekeeping genes are not stably expressed in situ[30][31] One example of metatranscriptomic application is in the study of the gut microbiome in inflammatory bowel disease.
[32] Several human genetic mutations have been linked to an increased susceptibility to IBD, but additional factors are needed for the full development of the disease.
In addition, the gut microbiome composition presents a high variability over time among people, with more pronounced variations in patient with IBD.
[33] These altered expression profiles are potentially the result of changes in the gut environment in patients with IBD, which include increased levels of inflammation, higher concentrations of oxygen and a diminished mucous layer.
[36] Metatranscriptomics has the advantage of allowing researchers to skip the assaying of biochemical products in situ (like mucus or oxygen) and enables evaluation of effects of environmental changes on microbial expression patterns in vivo for large human populations.
Examples of techniques applied: Microarrays: allow the monitoring of changes in the expression levels of many genes in parallel for both host and pathogen.