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. 2020 Nov 4;12:167. doi: 10.1186/s13148-020-00960-z

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 1 — The study and analysis workflow for ME/CFS patients and age-matched healthy control methylome: DNA was isolated from the Peripheral blood mononuclear cells (PBMCs) of 10 ME/CFS patients and 10 age/sex matched healthy controls. DNA was processed to produce reduced representation bisulphite sequencing libraries. This DNA was then sequenced and the data aligned and trimmed using Bismark. The aligned RRBS sequence data were then analyzed using two parallel analyses pipelines: DMAP and MethylKit. DMAP utilised an ANOVA F test comparing patients and controls (requiring at least seven from each group to be present in each comparison between fragments). Associated promoter and gene regions were then identified. Methylkit analysis pooled the patient and control groups before comparison using a Fisher’s test to identify differentially methylated cytosines (requiring at least seven from each group to be present in the comparison between cytosines). Promoter and gene regions were then isolated and investigated