Object's details: Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks

Provider:Repozytorium Uniwersytetu Łódzkiego

Description

Title:
Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks
Creator:
Pośpiech, Ewelina;
Pisarek, Aleksandra;
Rudnicka, Joanna;
Noroozi, Rezvan;
Boroń, Michał;
Masny, Aleksander;
Wysocka, Bożena;
Migacz-Gruszka, Kamila;
Lisman, Dagmara;
Pruszkowska-Przybylska, Paulina;
Kobus, Magdalena;
Szargut, Maria;
Dowejko, Joanna;
Stanisz, Kamila;
Zacharczuk, Julia;
Zieliński, Piotr;
Sitek, Aneta;
Ossowski, Andrzej;
Spólnicka, Magdalena;
Branicki, Wojciech
Contributor:
Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland;
Department of Forensic Genetics, Pomeranian Medical University in Szczecin, Szczecin, Poland;
Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Krakow, Poland;
Doctoral School of Exact and Natural Sciences, Jagiellonian University, Krakow, Poland;
Central Forensic Laboratory of the Police, Warsaw, Poland;
Department of Dermatology, Collegium Medicum of the Jagiellonian University, Krakow, Poland;
Department of Anthropology, Faculty of Biology and Environmental Protection, University of Łódź, Łódź, Poland;
Institute of Biological Sciences, Faculty of Biology and Environmental Sciences, Cardinal Stefan Wyszynski University in Warsaw, Warsaw, Poland;
Institute of Environmental Sciences, Faculty of Biology, Jagiellonian University, Krakow, Poland;
Center for Forensic Science, University of Warsaw, Warsaw, Poland;
Institute of Forensic Research, Krakow, Poland
Description:
Background: DNA methylation analysis has proven to be a powerful tool for age assessment. However, the implementation of epigenetic age prediction in diagnostics or routine forensic casework requires appropriate laboratory methods. In this study, we aimed to compare the performance of large-scale DNA methylation analysis protocols that show promise in terms of accuracy, throughput, multiplexing capacity, and high sensitivity. Results: The protocols were designed to target a predefined panel of 161 genomic CG/CA sites from four known estimators of epigenetic age-related parameters, optimized and validated using artificially methylated controls or blood samples. We successfully targeted 96% of these loci using two enrichment protocols: Ion AmpliSeq™, an amplicon-based method integrated with Ion Torrent S5, and SureSelectXT Methyl-Seq, a hybridization-based method followed by MiSeq FGx sequencing. Both protocols demonstrated high accuracy and robustness. Although hybridization assays have greater multiplexing capabilities, the best overall performance was observed for the amplicon-based protocol with the lowest variability in DNA methylation at 25 ng of starting DNA, mean observed marker coverage of ~ 6.7 k reads, and accuracy of methylation quantification with a mean absolute difference between observed and expected methylation beta value of 0.054. The Ion AmpliSeq method correlated strongly with genome-scale EPIC microarray data (R = 0.91) and showed superiority in terms of methylation measurement accuracy. Method-to-method bias was accounted for by the use of linear transformation, which provided a highly accurate prediction of calendar age with a mean absolute error of less than 5 years for the VISAGE and Hannum age clocks used. The pace of aging (PoAm) and the mortality risk score (MRS) estimators included in our panel represent next-generation clocks, were found to have low to moderate correlations with the VISAGE and Hannum models (R < 0.75), and thus may capture different aspects of epigenetic aging. Conclusions: We propose a laboratory tool that allows the quantification of DNA methylation in cytosines underlying four different clocks, thus providing broad information on epigenetic aging while maintaining a reasonable number of CpG markers, opening the way to a wide range of applications in forensics, medicine, and healthcare.;
This work is financed by the National Centre for Research and Development (NCBR), Poland, within the framework of call 10/2019 related to scientific research and studies for national defense and security [project no. DOB-BIO10/06/01/2019].
Object availability:
free access
Rights:
Uznanie autorstwa 4.0 Międzynarodowe;
http://creativecommons.org/licenses/by/4.0
Date:
2023.09.04;
2023
Type:
Article
Language:
English
Format:
application/pdf
Publisher:
Springer Nature
Relation:
Clinical Epigenetics;128;
15
Subject:
DNA methylation analysis methods;
High-throughput sequencing;
Target enrichment protocols;
Epigenetic age estimation;
Pace of aging;
Mortality risk score
Identifier:
Pośpiech, E., Pisarek, A., Rudnicka, J. et al. Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks. Clin Epigenet 15, 128 (2023). https://doi.org/10.1186/s13148-023-01545-2;
1868-7083;
http://hdl.handle.net/11089/47851 ;
10.1186/s13148-023-01545-2
Data provider:
Repozytorium Uniwersytetu Łódzkiego

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Object's details: Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks

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