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

Florian Trigodet, Karen Lolans, Emily Fogarty, Alon Shaiber, Hilary G. Morrison, Luis Barreiro, Bana Jabri, A. Murat Eren

Research output: Contribution to journalArticlepeer-review

24 Scopus citations


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. The 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 column-based kits with enzyme supplementation, rather than PC methods, may be more appropriate for long-read sequencing of metagenomes.

Original languageEnglish
Pages (from-to)1786-1802
Number of pages17
JournalMolecular Ecology Resources
Issue number5
StatePublished - Jul 2022
Externally publishedYes


  • high-molecular-weight DNA
  • long-read sequencing
  • metagenomics
  • nanopore


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