Open AccessExtraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling
Author(s) -
Federico Rossi,
Alessandro Crnjar,
Federico Comitani,
Rodrigo P. Feliciano,
Leonie Johanna Jahn,
George Malim,
Laura Southgate,
Emily Kay,
Rebecca J. Oakey,
Richard J. A. Buggs,
Andy Moir,
Logan Kistler,
Ana RodriguezMateos,
Carla Molteni,
Reiner Schulz
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0254971
Subject(s) - dna methylation , epigenetics , bisulfite sequencing , biology , quercus robur , genome , methylated dna immunoprecipitation , dna sequencing , bisulfite , computational biology , ancient dna , genetics , dna , gene , botany , medicine , gene expression , population , environmental health
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 whole-genome 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.