High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes

• Published in: bioRxiv
• Main Authors: Trigodet, Florian, Lolans, Karen, Fogarty, Emily, Shaiber, Alon, Morrison, Hilary G, Barreiro, Luis, Jabri, Bana, Eren, A Murat
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 03.03.2021; Cold Spring Harbor Laboratory
• Edition: 1.2
• Subjects: Agarase; Chloroform; Conflicts of interest; Deoxyribonucleic acid; DNA; DNA sequencing; Genomes; Microbiology; Molecular weight; Size distribution; high-molecular-weight DNA; nanopore; long-read sequencing; metagenomics
• ISSN: 2692-8205; 2692-8205
• DOI: 10.1101/2021.03.03.433801

Abstract By offering extremely long contiguous characterization of individual DNA molecules, rapidly emerging long-read sequencing strategies offer comprehensive insights into the organization of genetic information in genomes and metagenomes. However, successful long-read sequencing experiments demand high concentrations of highly purified DNA of high molecular weight (HMW), which limits the utility of established DNA extraction kits designed for short-read sequencing. Challenges associated with input DNA quality intensify further when working with complex environmental samples of low microbial biomass, which requires new protocols that are tailored to study metagenomes with long-read sequencing. Here, we use human tongue scrapings to benchmark six HMW DNA extraction strategies that are based on commercially available kits, phenol-chloroform (PC) extraction, and agarose encasement followed by agarase digestion. A typical end goal of HMW DNA extractions is to obtain the longest possible reads during sequencing, which is often achieved by PC extractions as demonstrated in sequencing of cultured cells. Yet our analyses that consider overall read-size distribution, assembly performance, and the number of circularized elements found in sequencing results suggest that non-PC methods may be more appropriate for long-read sequencing of metagenomes. Competing Interest Statement The authors have declared no competing interest. Footnotes * Conflict of interest The authors declare no conflict of interest