Accel ngs methyl seq dna library kit with unique dual indexing

The quality of extracted cfDNA was analyzed by cfDNA ScreenTape analysis on the 4150 TapeStation System (Agilent Technologies) prior to bisulfite conversion. The cfDNA was then processed with EZ DNA methylation-gold kit (Zymo Research) as per the manufacturer’s recommendation to convert unmethylated cytosine residues to uracil. The methyl-Seq DNA library was prepared using the Accel-NGS Methyl-Seq DNA Library Kit with Unique Dual Indexing (Swift Biosciences) for whole-genome bisulfite sequencing. The quality of the constructed DNA library was assessed using a high-sensitivity D1000 ScreenTape and quantified by the Quant-iT PicoGreen dsDNA assay kit (Life Technologies). The DNA libraries were then pooled in equimolar concentrations and subjected to 2 × 100 bp paired-end DNA sequencing on the Illumina NovaSeq 6000 platform.

Size distribution of the cfDNA was determined using Cell-free DNA ScreenTape assay on Agilent 4150 TapeStation System according to the manufacturer’s instructions to assure the absence of contamination with genomic DNA. An input of 5 – 50 ng of isolated cfDNA, depending on the availability, was used to perform bisulfite conversion using EZ DNA methylation-Gold kit (Zymo Research) as per the manufacturer’s recommendation. Libraries were prepared using the Accel-NGS Methyl-Seq DNA Library Kit with Unique Dual Indexing (Swift Biosciences) for whole-genome bisulfite sequencing according to the manufacturer’s instructions. The quality of the constructed cfDNA library was visualized using a high-sensitivity D1000 ScreenTape and quantified using the Quant-iT PicoGreen dsDNA Assay kit (Life Technologies). The DNA libraries were then normalized in equimolar concentrations and were subjected for ~200 M reads by 2X100bp, paired-end DNA sequencing on the Illumina NovaSeq 6000.