JHSC-0270_RRBS
RRBS library was created using the Ovation RRBS Methyl-Seq kit (Tecan Group Ltd., Zurich, Switzerland). To account for the highly degraded DNA from FFPE and plasma samples, the material was first treated with one unit of Shrimp Alkaline Phosphatase (New England Biolabs, Ipswich, MA) to remove phosphorylated DNA which might interfere with downstream analysis. Briefly, 0.1 – 100ng of genomic DNA was digested using MspI, and Illumina-compatible cytosine-methylated adaptor were ligated to the enzyme-digested DNA. For lower concentrations of DNA, adapters were diluted 1:40 to 1:120, in order to decrease the representation of randomly fragmented DNA and adapter-dimers in the final library. RRBS libraries were then visualized using Bioanalyzer High Sensitivity DNA chips (Agilent, Santa Clara, CA), and those passing QC were subsequently sequenced as 100bp paired-end reads on an Illumina NovaSeq instrument with a target sequencing depth of 300M PE reads (150M clusters). After sequencing, Fastq files were obtained and adapters were trimmed using trimmomatic. Alignment and retrieval of DNA Methylation (in percent of total methylated Cytosines) was performed using Bismark v 0.22 against the GRCh38 human genome. Samples with < 50% mapping rate and, < 60M aligned reads were excluded from further analysis. Finally, cytosines with coverage < 10 were filtered out to assure high confidence DNA Methylation analysis.