Targeted DNA Methylation Sequencing

MeD-seq assays were essentially performed as previously described [16 (link)]. In short, 8 µl genomic DNA (input ranged from 117 to 1728 ng) from frozen tissues, specified amounts of MCF7 genomic DNA or plasma-derived cfDNA were digested with LpnPI (New England Biolabs, Ipswich, MA) yielding 32 bp fragments around the fully methylated recognition site containing a CpG. Samples were prepped for sequencing using the ThruPLEX DNA-seq 96D kit (Rubicon Genomics, Takara Bio Europe, Saint-Germain-en-Laye, France) and purified on a Pippin HT system with 3% agarose gel cassettes (Sage Science, Beverly, MA). Libraries were multiplexed and sequenced on an Illumina HiSeq 2500 for 50 bp single reads according to the manufacturer's instructions (Illumina, San Diego, CA). Samples were first sequenced until ~ 2 M reads and continued to a total of ~ 20 M reads only when the fraction of reads that passed the LpnPI filter (explained below) was at least 20%.