Targeted bisulfite sequencing: A novel tool for the assessment of DNA methylation with high sensitivity and increased coverage. (October 2020)

• Record Type: Journal Article
• Authors: Moser, D.A.; Müller, S.; Hummel, E.M.; Limberg, A.S.; Dieckmann, L.; Frach, L.; Pakusch, J.; Flasbeck, V; Brüne, M.; Beygo, J.; Klein-Hitpass, L.; Kumsta, R.
• Is Part Of: Psychoneuroendocrinology. Volume 120(2020)
• Publication Date: 2020-10
• Subjects: DNA methylation -- Epigenetics -- Targeted bisulfite sequencing -- Borderline personality disorder (BPD); Psychoneuroendocrinology -- Periodicals; Endocrinology -- Periodicals; Neurology -- Periodicals; Psychiatry -- Periodicals; Neuropsychoendocrinologie -- Périodiques; 616.8
• DOI: 10.1016/j.psyneuen.2020.104784
• Languages: English
• ISSNs: 0306-4530

Abstract

Highlights: Targeted bisulfite sequencing facilitates DNA methylation analysis and represents a sensitive and cost efficient tool for epigenetic analysis. The method shows excellent reliability and validity compared to other methods. This highly flexible NGS-based approach can be used for targeted analysis of selected candidate genes and for validation of hits derived from whole-genome or array-based approaches. It enables high-throughput analysis and offers the possibility of parallel investigation of many candidate genes in the same run. We expect this method to close the gap between coverage and precision in epigenetic research of stress-associated phenotypes. Abstract: DNA methylation analysis is increasingly used in stress research. Available methods are expensive, laborious and often limited by either the analysis of short CpG stretches or low assay sensitivity. Here, we present a cost-efficient next generation sequencing-based strategy for the simultaneous investigation of multiple candidate genes in large cohorts. To illustrate the method, we present analysis of four candidate genes commonly assessed in psychoneuroendocrine research: Glucocorticoid receptor ( NR3C1), Serotonin transporter (SLC6A4), FKBP Prolyl isomerase 5 (FKBP5), and the Oxytocin receptor ( OXTR). DNA methylation standards (100 %; 75 %; 50 %; 25 % and 0 %) and DNA of a female and male donor were bisulfite treated in three independent trials and were used to generate sequencing libraries for 42 CpGs from the NR3C1 1 F promoter region, 84 CpGs of the SLC6A4 5′ regulatory region, 5 CpGs located in FKBP5 intron 7, and additional 12 CpGs located in a potential enhancer element in intron 3 of the OXTR . In addition, DNA of 45 patients with borderline personality disorder (BPD) and 45 healthy controls was assayed. Multiplex libraries of all samples were sequenced on a MiSeq system and analyzed for mean methylation values of all CpG sites using amplikyzer2 software. Results indicated excellent accuracy of the assays when investigating replicates generated from the same bisulfite converted DNA, and very high linearity (R 2 > 0.9) of the assays shown by the analysis of differentially methylated DNA standards. Comparing DNA methylation between BPD and healthy controls revealed no biologically relevant differences. The technical approach as described here facilitates targeted DNA methylation analysis and represents a highly sensitive, cost-efficient and high throughput tool to close the gap between coverage and precision in epigenetic research of stress-associated phenotypes.