Metagenomics versus total RNA sequencing: most accurate data-processing tools, microbial identification accuracy and perspectives for ecological assessments

• Published in: Nucleic acids research 2022-09, Vol.50 (16), p.9279-9293
• Main Authors: Hempel, Christopher A, Wright, Natalie, Harvie, Julia, Hleap, Jose S, Adamowicz, Sarah J, Steinke, Dirk
• Format: Article
• Language: English
• Subjects: Genomics; High-Throughput Nucleotide Sequencing - methods; Metagenomics - methods; Microbiota - genetics; RNA, Ribosomal, 16S - genetics; Sequence Analysis, RNA
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkac689

Abstract Metagenomics and total RNA sequencing (total RNA-Seq) have the potential to improve the taxonomic identification of diverse microbial communities, which could allow for the incorporation of microbes into routine ecological assessments. However, these target-PCR-free techniques require more testing and optimization. In this study, we processed metagenomics and total RNA-Seq data from a commercially available microbial mock community using 672 data-processing workflows, identified the most accurate data-processing tools, and compared their microbial identification accuracy at equal and increasing sequencing depths. The accuracy of data-processing tools substantially varied among replicates. Total RNA-Seq was more accurate than metagenomics at equal sequencing depths and even at sequencing depths almost one order of magnitude lower than those of metagenomics. We show that while data-processing tools require further exploration, total RNA-Seq might be a favorable alternative to metagenomics for target-PCR-free taxonomic identifications of microbial communities and might enable a substantial reduction in sequencing costs while maintaining accuracy. This could be particularly an advantage for routine ecological assessments, which require cost-effective yet accurate methods, and might allow for the incorporation of microbes into ecological assessments.