Computational protocol: Accumulated promoter methylation as a potential biomarker for esophageal cancer

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Protocol publication

[…] PCR amplicons were then diluted and amplified using the indexed primers. Specifically, a 20 μl mixture was prepared for each reaction, including 1x buffer (NEB, MA, USA), 0.3 mM dNTP, 0.3 μM forward primer, 0.3 μM index primer, 1 U Q5TM DNA polymerase (NEB, MA, USA) and 1 μL of diluted template (PCR amplicons from the previous step). The cycling program was 98°C for 30 sec; 11 cycles of 98°C for 10 sec, 65°C for 30 sec, 72°C for 30 sec; 72°C for 5 min. The PCR products (170 bp −270 bp) were separated by agarose electrophoresis and purified using the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). Libraries from different samples were quantified and pooled together, followed by sequencing on the Illumina MiSeq platform according to the manufacturer's protocols. Sequencing was performed with a 2 × 300 bp paired-end mode. Quality control of sequencing-reads was performed by FastQC ( Filtered-reads were aligned back to the reference genome using the Bismark software ( After reads recalibration withUSEARCH [], the methylation and haplotype were analyzed using the Perl script. [...] We used the IBM SPSS Statistics 19.0 (IBM Corp., NY, USA) and the R program ( to analyze the data. Individual and cumulative methylation statuses of candidate genes were analyzed. We used the t-test, ANOVA or nonparametric test to compare the differences of methylation between groups. Considering the false positive caused by multiple comparisons, the Bonferroni correction was applied. The receiver operative characteristics (ROC) curve was drafted to reflect the diagnostic value of biomarkers. The area under the curve (AUC) together with 95% confidence interval (CI) were calculated. […]

Pipeline specifications

Software tools FastQC, Bismark, SPSS
Applications Miscellaneous, BS-seq analysis
Organisms Homo sapiens
Diseases Esophageal Neoplasms, Neoplasms