Benchmarking DNA extraction protocols across use cases for culture-independent Nanopore metagenomics

Oxford Nanopore Technologies (ONT) sequencing offers several advantages for metagenomics, including long reads, rapid turnaround, low upfront cost, scalability and portability. However, for ONT metagenomics, DNA yield, quality and integrity are important considerations when selecting an extraction method. Many metagenomic extraction methods use harsh lysis conditions to extract a wide range of species and provide an accurate community composition, but these conditions can compromise DNA fragment length. Therefore, extraction methods for ONT metagenomics must balance DNA shearing and recovery with representative community lysis. We systematically evaluated DNA extraction methods for ONT metagenomic sequencing using a use case-oriented framework. Among nearly 50 extraction methods screened, 7 were selected for detailed comparison based on suitability for metagenomics, variation in methodology, availability, cost and processing time: Norgen BioTek Corp's Stool DNA Isolation (NG), Zymo Research's ZymoBIOMICS Quick-DNA HMW MagBead (ZMG), Qiagen's DNeasy Blood and Tissue (QBT), Macherey-Nagel's NucleoMag DNA Microbiome (MN), Zymo Research's ZymoBIOMICS DNA Mini Prep (ZMI), Qiagen's DNeasy PowerSoil/QIAamp PowerFecal Pro (PS) and Qiagen's QIAamp Fast DNA Stool Mini (QIA). Methods were tested using Zymo Research's ZymoBIOMICS Microbial Community Standard (MCS), a matrix-free mock community with known composition. DNA extracts were sequenced on an ONT PromethION using the Rapid Barcoding Kit, except QIA due to insufficient DNA yield. Metrics for the method, DNA extracts, sequencing and genomes were evaluated, revealing trade-offs between methods. The two magnetic bead methods, MN and ZMG, produced the highest mean read length N50 values (13.9 and 16.5 kb, respectively) but showed apparent community compositions skewed towards Gram-negative bacteria. In contrast, ZMI and PS maintained a community composition close to expected, with reduced mean read length N50 values (4.5 vs. 7.5 kb). Performance across various metrics is presented in the context of the following use cases: maximizing genome coverage and assembly completeness, preserving composition accuracy, targeting specific species and limiting required resources (equipment, time or budget). The metrics and use case considerations presented offer practical guidance for informed selection of DNA extraction methods for ONT metagenomics. For accurate community composition, ZMI or PS are recommended, while PS and ZMG perform best at maximizing genome coverage and assembly completeness. NG and QBT may be the most economical options, though performance trade-offs were observed. Finally, PS may be the preferred method for time-sensitive diagnostic or field applications.