Comparison of Metatranscriptomics and Targeted-sequencing Approaches for Comprehensive Microbiome Profiling

Abstract Targeted-sequencing approaches, such as 16S gene profiling, viral metagenomics, and human mRNA sequencing are commonly performed for the exploration of the microbiome, yet their combination can be expensive and time-consuming. Metatranscriptomics provides a snapshot of the entire active microbiome trough bulk RNA sequencing in a single test, but lacks adequate comparisons with targeted-sequencing approaches. We compared metatranscriptomics and targeted sequencing methods focusing on bacterial, viral, and human components, from 20 nasopharyngeal aspirates from infants under 1 year old and hospitalized for bronchiolitis at the Hospices Civils de Lyon. RNA microbiome concordance reached 86% and 78% for RNA viruses and human coding genes, respectively. Patient clustering was comparable regarding 2650 host transcripts sequenced with metatranscriptomics and mRNA-Seq. Metatranscriptomics detected RNA of eukaryotic and prokaryotic DNA viruses, indicating potential for discerning replicative from latent DNA microbiome. Transcriptionally active bacteriome corresponded to 82% of bacteria exceeding 0.5% relative abundance, showing different transcriptional profiles depending on bacterial species. Multi-omics technologies enhance epidemiology, investigate trans-kingdom interactions, and provide opportunities to establish microbiome biomarkers. With sufficient depth of sequencing, metatranscriptomics complements and aligns with various aspects of targeted-sequencing approaches. Further clinical studies are essential to establish the position of metatranscriptomics in critical acute situations and cases of diagnostic uncertainty.