Multi-omics analyses from a single sample: Prior metabolite extraction does not alter the 16S rRNA-based characterization of prokaryotic community in a diversity of sample types

Abstract

Massive sequencing of the 16S rRNA gene has become a standard first step to describe and compare microbial communities from various samples. Parallel analysis of high numbers of samples makes it relevant to the statistical testing of the influence of natural or experimental factors and variables. However, these descriptions fail to document changes in community or ecosystem functioning. Non-targeted metabolomics are a suitable tool to bridge this gap, yet extractions protocols are different. In this study, prokaryotic community compositions are documented by 16S rRNA sequencing after direct DNA extraction, or after metabolites extraction followed by DNA extraction. Results obtained using the V3-V4 region on non-axenic cultures of cyanobacteria, lake water column, biofilm, gut of wild and lab-reared fish, indicate that prior extraction of metabolites does not influence the obtained image of prokaryotic communities. This validates sequential extraction of metabolites followed by DNA as a way to combine 16S rRNA sequencing with metabolome characterization from a single sample. This approach has the potential to complement community structure characterization with a proxy of their functioning, without the uncertainties associated with the use of separate samples.

New sensitive tools to characterize meta-metabolome response to short- and long-term cobalt exposure in dynamic river biofilm communities

“…The comparison of variations at different levels of biological response through different omic approaches (Fig. 6) underlined the value of performing first metabolite extraction prior to DNA extraction on the same sample (Duperron et al, 2023) as applied here for ecotoxicological investigations. Indeed, the three parameters studied (metabolites, 16S and 18S ASVs) came from the same samples, and provided a good representation of the response of microbial communities to contamination on individualized biofilm samples.…”

“…Subsequently to methanol extraction of metabolites, (described below in section 2.5), DNA was extracted from the pellets of the same samples (Duperron et al, 2023) using the QIAGEN DNeasy® PowerSoil® Pro Kit DNA extraction kit following the manufacturer's instruction. The V4-V5 region of the 16S rRNA encoding gene was amplified using primers 515F (5'-GTGYCAGCMGCCGCGGTA-3') and 928R (5'-ACTYAAAKGAATTGRCGGGG-3') (Wang and Qian, 2009).…”