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Extraction and high-throughput sequencing of oak heartwood DNA: assessing the feasibility of genome-wide DNA methylation profiling

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Federico Rossi, Alessandro Crnjar, Rodrigo Feliciano, Federico Comitani, Leonie Jahn, George Malim, Laura Southgate, Emily Kay, Rebecca Oakey, Richard Buggs, Andy Moir, Logan Kistler, Ana Maria Rodriguez Mateos, Carla Molteni, Reiner Schulz

Original languageEnglish
Article numbere0254971
JournalPLoS One
Volume16
Issue number11
Early online date18 Nov 2021
DOIs
Accepted/In press27 Oct 2021
E-pub ahead of print18 Nov 2021
Published18 Nov 2021

Bibliographical note

Publisher Copyright: © 2021 Rossi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

King's Authors

Abstract

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of wholegenome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

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