Optimizing storage, high-molecular weight DNA extraction and genome reconstructions from human faecal samples

Abstract Metagenomic sequencing has become an essential tool for assessing the composition and functional potential of microbial communities. However, established metagenomic DNA extraction protocols are limited by trade-offs between universal taxonomic representation, DNA quality, quantity and molecule length, making them mostly unsuitable for 3 rd generation sequencing. Methods We assessed the effectiveness of high molecular weight (HMW) DNA workflows from faecal samples, using different combinations of collection, storage and extraction kits. Faecal samples were either immediately processed, frozen at -80°C or stored in DNA/RNA Shield, GutAlive or OMNIgeneGUT and then subjected to three different DNA isolation kits, each modified for mechanical, chemical and enzymatic cell lysis steps, totalling 32 evaluated combinations. Isolated DNA was assessed for its quality and quantity, while taxonomic consistency was evaluated using metataxonomics and validated with metagenomic sequencing. Results The yield of HMW DNA differed substantially between storage and DNA extraction protocols. Specifically, storage protocol had the highest impact on HMW DNA recovery, with DNA/RNA Shield storage yielding on average 51% more HMW DNA >30kb. Similarly, the sample storage had independently the effect size of R 2 = 26.9% on the microbiome composition, and since the DNA extraction protocol in our study inherently combines both DNA extraction kit and lysis type, the latter had the largest effect size ( R 2 = 12.8%). While all kits had specific dis/advantages and biases, DNA/RNA Shield and Maxwell RSC PureFood GMO and Authentication kit performed best overall for long-read metagenomics. The protocol was validated on a) isolate genomes and b) 4 faecal samples, demonstrating both increased DNA yield at >30kb molecule length. Using the MG-TK pipeline these metagenomes yielded genomes on average, per sample, 48 Pacific Biosciences (PacBio) and 55 Oxford Nanopore Technologies (ONT) circular bacterial metagenomic assembled genomes. Conclusion The current gold-standard of freezing faecal samples was surprisingly inconsistent and inefficient for long-read metagenomics, necessitating rethinking gut microbial study designs. We provide a simple and cheap protocol that preserves both taxonomic composition and HMW DNA quality, while our bioinformatics workflow allows for complete bacterial genome reconstruction.