Comparative evaluation of DNA extraction protocols for neonatal gut microbiota profiling in a resource-limited setting

Accurate and reproducible gut microbiome profiling depends heavily on the DNA extraction method, particularly in low-biomass samples such as neonatal stool. In this study, we evaluated the performance of three commercially available DNA extraction kits - QIAamp Fast DNA Stool Mini, DNeasy PowerSoil Pro, and ZymoBIOMICS DNA Miniprep on neonatal stool samples collected in a resource-limited hospital in Kano, Nigeria. Samples were stored under various conditions (temperature and preservatives), and DNA was extracted and sequenced using Oxford Nanopore long-read technology. Our results showed that fresh, unpreserved samples yielded significantly higher DNA concentrations than stored or preserved samples. Kits incorporating mechanical lysis (PowerSoil and ZymoBIOMICS) outperformed the chemical lysis-based QIAamp kit in DNA yield and microbial recovery. While microbial diversity and taxonomic profiles were comparable between PowerSoil and ZymoBIOMICS, PowerSoil produced longer sequencing reads and required less processing time, making it more suitable for long-read metagenomics in field conditions. We conclude that same-day processing using bead-beating-based extraction kits improves yield and reduces bias in neonatal gut microbiome studies. These findings are especially relevant for low-resource settings, where equipment limitations and delayed sample processing can impact data quality and study scalability.