Comparison of Metagenomics and Metatranscriptomics Tools: A Guide to Making the Right Choice

Abstract

The study of microorganisms is a field of great interest due to their environmental (e.g., soil contamination) and biomedical (e.g., parasitic diseases, autism) importance. The advent of revolutionary next-generation sequencing techniques, and their application to the hypervariable regions of the 16S, 18S or 23S ribosomal subunits, have allowed the research of a large variety of organisms more in-depth, including bacteria, archaea, eukaryotes and fungi. Additionally, together with the development of analysis software, the creation of specific databases (e.g., SILVA or RDP) has boosted the enormous growth of these studies. As the cost of sequencing per sample has continuously decreased, new protocols have also emerged, such as shotgun sequencing, which allows the profiling of all taxonomic domains in a sample. The sequencing of hypervariable regions and shotgun sequencing are technologies that enable the taxonomic classification of microorganisms from the DNA present in microbial communities. However, they are not capable of measuring what is actively expressed. Conversely, we advocate that metatranscriptomics is a “new” technology that makes the identification of the mRNAs of a microbial community possible, quantifying gene expression levels and active biological pathways. Furthermore, it can be also used to characterise symbiotic interactions between the host and its microbiome. In this manuscript, we examine the three technologies above, and discuss the implementation of different software and databases, which greatly impact the obtaining of reliable results. Finally, we have developed two easy-to-use pipelines leveraging Nextflow technology. These aim to provide everything required for an average user to perform a metagenomic analysis of marker genes with QIMME2 and a metatranscriptomic study using Kraken2/Bracken.

First report on metagenomics and their predictive functional analysis of fermented bamboo shoot food of Tripura, North East India

“…The putative microbial population reads from six samples were classified using Kraken2 against the reference database containing all Refseq bacterial, archaeal, fungal, and viral genomes with a 0.1 confidence threshold ( Marcelino et al, 2020 ). After the classification by Kraken2, Bracken was used to re-estimate the different microbial abundances at taxa levels from species to phylum using a read length parameter of 150 ( Terrón-Camero et al, 2022 ).…”

Unveiling the Human Gastrointestinal Tract Microbiome: The Past, Present, and Future of Metagenomics

“…The second step of metatranscriptomics analysis comprises the analysis of the generated data. Although a number of bioinformatics algorithms have been developed for the study of the metagenome, the design of computational methods for analyzing metatranscriptomic data remains a challenging process [ 129 ]. It should be mentioned that, except for the NGS platforms that have already been established in the field of microbiology for the study of metatranscriptome through RNA-seq, the brand-new direct RNA nanopore sequencing approach constitutes a highly promising strategy.…”

Gut Dysbiosis and Blood-Brain Barrier Alteration in Hepatic Encephalopathy: From Gut to Brain

“…Each approach images different types of biological information from the microbiota communities. Current investigations combine more than one omics approach to image a highresolution picture of the gut microbiota dynamics [41]. Genomics is concerned with identifying the genetic composition of a single bacterium, but metagenomics is concerned with identifying the genetic composition of an entire community of bacterial communities (for instance, the entire gut microbiome of humans) [42].…”