Nebnext enzymatic methyl seq em seq kit

Libraries were prepared with 1, 2, 5 or 10ng DNA using the NEBNext® Enzymatic Methyl-seq (EM-seq™) kit and Unique Dual Index Primer pairs (New England Biolabs) input according to manufacturer’s instructions (NEB #E7120 S/L v6.0_3/2). Unmethylated lambda phage DNA and plasmid pUC19 DNA, methylated at CpG sites, were added as negative and positive controls, respectively. A total of 12 amplification cycles were used for 1ng of input DNA and 10 cycles for other input amounts. Libraries were pooled equimolarly and then sent for sequencing.

Urine cell-free DNA (cfDNA) extracted from urine supernatant samples of ovarian cancer patients was further characterized by shallow whole-genome sequencing (~1× coverage). The cfDNA was quantified and analyzed using a Cell-free DNA ScreenTape assay of the Agilent 4200 TapeStation System (Agilent) for quality control before sequencing. Sequencing libraries of the first pilot series of urine supernatant DNA were prepared using the ThruPLEX Plasma-seq Kit (Takara Bio, Mountain View, CA, USA) for whole-genome sequencing according to the manufacturer’s instructions. The remaining samples were prepared using the NEBNext® Enzymatic Methyl-seq (EM-seq) Kit (NEB, Ipswich, MA, USA). EM-seq was performed according to manufacturer’s guidelines for standard insert libraries with 14 PCR cycles. Libraries were quantified and quality-checked using the D1000 ScreenTape Analysis Assay (Agilent) before pooling. Paired-end 150 base pair (bp) libraries were pooled in equimolar amounts and sequenced on a NovaSeq6000 (Illumina) (GenomeScan, Leiden). The processing of sequencing data and subsequent analysis of SCNA and cfDNA fragmentation patterns are provided in the Supplemental Methods. Shallow whole-genome sequencing of paired FFPE primary tumor tissue was performed to verify copy number profile concordance and is also described in the Supplemental Methods.