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High molecular weight DNA extraction strategies for long-read sequencing of complex metagenomes.
| Content Provider | Europe PMC |
|---|---|
| Author | Trigodet, Florian Lolans, Karen Fogarty, Emily Shaiber, Alon Morrison, Hilary G. Barreiro, Luis Jabri, Bana Eren, A. Murat |
| Abstract | AbstractBy 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. |
| Related Links | https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC9177515&blobtype=pdf |
| Page Count | 17 |
| ISSN | 1755098X |
| Journal | Molecular Ecology Resources [Mol Ecol Resour] |
| Volume Number | 22 |
| DOI | 10.1111/1755-0998.13588 |
| PubMed Central reference number | PMC9177515 |
| Issue Number | 5 |
| PubMed reference number | 35068060 |
| e-ISSN | 17550998 |
| Language | English |
| Publisher | John Wiley and Sons Inc. |
| Publisher Date | 2022-02-07 |
| Publisher Place | Hoboken |
| Access Restriction | Open |
| Rights License | This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. © 2022 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd. |
| Subject Keyword | high‐molecular‐weight DNA long‐read sequencing metagenomics nanopore |
| Content Type | Text |
| Resource Type | Article |
| Subject | Genetics Ecology, Evolution, Behavior and Systematics Biotechnology |
