Metatranscriptomics

Metatranscriptomics allows the analysis of messenger RNAs expressed within complex microbial communities, revealing functional activity rather than simply the genetic presence of microorganisms (Zang et al., 2021).

Experimental steps

a) Collection & extraction, sampling respecting rapid RNA stabilisation to prevent degradation. Extraction of total RNA from the sample (soil, water, intestinal flora, etc.), which mainly contains rRNA.

b) Depletion of rRNA (rRNA/tRNA) using commercial kits or bead capture, eliminating up to 70–85% of rRNA.

c) Fragmentation & library creation

Conversion of mRNA to cDNA by reverse transcription with strand-specific protocols, often for Illumina platforms offering high-throughput short reads (≥100 million reads per sample), library preparation: adapter ligation, PCR amplification.
Another option: direct RNA sequencing by nanopores (Oxford Nanopore), long reads, without PCR, without assembly bias, but with a higher error rate.

d) High-throughput sequencing, Illumina, Element BioSciences or MGI for short reads; Oxford Nanopore for long reads, providing access to complete transcripts or isoforms.

e) Bioinformatic analysis

Quality control & filtering, using tools such as FastQC, Trimmomatic, SortMeRNA to remove low-quality reads or those derived from rRNA/tRNA.
Assembly/mapping, assembly of reads (de novo or by mapping to reference databases), taxonomic assignment using tools such as Kraken2, BLASTN, mapping to SILVA, Greengenes or genomic databases.
Functional annotation, identification of ORFs, annotation via KEGG, MetaHIT, M5nr, COG, GO, VFDB or CARD databases depending on the request (antibiotic resistance, virulence, etc.) → assignment of biological functions.

New technique: Use of direct RNA sequencing by nanopore to access long transcripts, isoforms and structural RNAs without cDNA, currently being optimised.

Overview of the metatranscriptomics analysis workflow. First step after RNA extraction is ribosomal RNA (rRNA) depletion leading to an enrichment of messenger RNA (mRNA), thereby improving the detection and analysis of gene expression. The purified mRNA is then reverse transcribed into complementary DNA (cDNA), a process that converts RNA into a stable DNA form. Subsequently, the cDNA undergoes high-throughput sequencing to produce short sequence reads. These reads are assembled into transcripts, which represent the expressed genes in the microbial community. Annotation tools are applied to these transcripts, assigning functional labels to elucidate their biological roles. Following annotation, statistical analyses such as differential expression analysis are employed to discern gene expression patterns. (Source: Esser et al., 2024)

Zhang Y, et al. Metatranscriptomics for the Human Microbiome and Microbial Community Functional Profiling, Annual Review of Biomedical Data Science Vol. 4:279-311 (juillet 2021) https://doi.org/10.1146/annurev-biodatasci-031121-103035

Esser M, Brinkmann M and Hecker M, Solving freshwater conservation challenges through next-generation sequencing approaches, DOI: 10.1039/D4VA00112E (Perspective) Environ. Sci.: Adv., 2024, 3, 1181-1196, https://pubs.rsc.org/en/content/articlehtml/2024/va/d4va00112e

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Update : July 2025

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