Microbiol Spectr. 2025 Oct 10. e0191225
Caitriona Brennan,
Justin P Shaffer,
Pedro Belda-Ferre,
Ipsita Mohanty,
Yuhan Weng,
Kalen Cantrell,
Gail Ackermann,
Celeste Allaband,
MacKenzie Bryant,
Sawyer Farmer,
Antonio González,
Daniel McDonald,
Cameron Martino,
Michael J Meehan,
Gibraan Rahman,
Rodolfo A Salido,
Tara Schwartz,
Se Jin Song,
Caitlin Tribelhorn,
Helena M Tubb,
Pieter C Dorrestein,
Rob Knight.
An essential aspect of population-based research is collecting samples outside of a clinical setting. This is crucial because microbial populations are highly dynamic, varying significantly across hosts, environments, and time points, a variability that clinical sample collection alone cannot fully capture. At-home sample collection enables the inclusion of a larger and more diverse group of participants, accounting for differences in ethnicity, age, and other factors. However, managing large studies is challenging due to the complexities involved in sample acquisition, processing, and analysis. Building on our previous work demonstrating the effectiveness of single 1 mL barcoded, racked Matrix Tubes in reducing sample processing time and well-to-well contamination for paired DNA and metabolite extraction, we further validate this method against a previously benchmarked plate-based approach using the same extraction reagents. This validation focuses on samples from the built environment, human skin, human saliva, and feces from mice and humans. Importantly, we explore the impact of using a mix of bead sizes during bead-beating for cell lysis, demonstrating that it enhances taxonomic recovery compared to a single bead size. Finally, we assess the potential of 95% isopropanol for room-temperature sample preservation. Our results show that isopropanol performs comparably to 95% ethanol in many cases, suggesting it is viable as an alternative when ethanol is unavailable. Beyond minimizing contamination, halving processing time, eliminating human error during sample plating, and streamlining metadata curation, the Matrix tube approach produces metabolomic, 16S, and shotgun metagenomic data consistent with the Plate-based Method for both high- and low-biomass samples.
IMPORTANCE: Numerous studies have linked the microbiome to human and environmental health, yet many fundamental questions remain unanswered. Large-scale studies with robust statistical power are required to identify important covariates against a background of confounding factors. Cross-contamination, limited throughput, and human error have been identified as major setbacks when processing large numbers of samples. We present a streamlined method for sample accession and extraction of metabolites and DNA for both high- and low-biomass samples. This approach, previously shown to significantly reduce cross-contamination, employs an automation-friendly, single barcoded tube per sample. Additionally, we demonstrate that 95% isopropanol serves as an effective ambient-temperature storage solution for many sample types, providing an alternative in regions where ethanol is unavailable or restricted. This method has significant implications for the field, enabling large-scale studies to generate accurate insights with greater efficiency and expanded accessibility in situations in which ethanol is more costly or otherwise not available.
Keywords: 16S rRNA gene; automation; contamination; large-scale studies; metabolomics; metagenomics; microbiome; sample storage; study design