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Background  DNA methylation has been implicated as a promising biomarker for precise cancer diagnosis. However, limited DNA methylation-based biomarkers have been described in esophageal squamous cell carcinoma (ESCC).    Methods  A high-throughput DNA methylation dataset (100 samples) of ESCC from The Cancer Genome Atlas (TCGA) project was analyzed and validated along with another independent dataset (12 samples) from the Gene Expression Omnibus (GEO) database. The methylation status of peripheral blood mononuclear cells and peripheral blood leukocytes from healthy controls was also utilized for biomarker selection. The candidate CpG sites as well as their adjacent regions were further validated in 94 pairs of ESCC tumor and adjacent normal tissues from the Chinese Han population using the targeted bisulfite sequencing method. Logistic regression and several machine learning methods were applied for evaluation of the diagnostic ability of our panel.    Results  In the discovery stage, five hyper-methylated CpG sites were selected as candidate biomarkers for further analysis as shown below: cg15830431,  P  = 2.20 × 10 −4 ; cg19396867,  P  = 3.60 × 10 −4 ; cg20655070,  P  = 3.60 × 10 −4 ; cg26671652,  P  = 5.77 × 10 −4 ; and cg27062795,  P  = 3.60 × 10 −4 . In the validation stage, the methylation status of both the five CpG sites and their adjacent genomic regions were tested. The diagnostic model based on the combination of these five genomic regions yielded a robust performance (sensitivity = 0.75, specificity = 0.88, AUC = 0.85). Eight statistical models along with five-fold cross-validation were further applied, in which the SVM model reached the best accuracy in both training and test dataset (accuracy = 0.82 and 0.80, respectively). In addition, subgroup analyses revealed a significant difference in diagnostic performance between the alcohol use and non-alcohol use subgroups.    Conclusions  Methylation profiles of the five genomic regions covering cg15830431 ( STK3 ), cg19396867, cg20655070, cg26671652 ( ZNF418 ), and cg27062795 ( ZNF542 ) can be used for effective methylation-based testing for ESCC diagnosis.    Electronic supplementary material  The online version of this article (10.1186/s13148-017-0430-7) contains supplementary material, which is available to authorized users.

Targeted bisulfite sequencing identified a panel of DNA methylation-based biomarkers for esophageal squamous cell carcinoma (ESCC)