Repli g whole genome amplification kit

One hundred percent methylated DNA was commercially sourced from NEB (CpG Methylated Jurkat Genomic DNA, Cat# N4002S). Zero percent methylated DNA was created by performing whole-genome amplification of commercially bought human genomic DNA (Roche Human Genomic DNA Cat# 11691112001), using the Qiagen REPLI-g whole-genome amplification kit, according to the manufacturer’s instruction. Graduated methylated controls (that is, 25%, 50%, 75%) were made by determining the amount of amplifiable DNA in the 100% and 0% methylated DNA samples using qPCR and pooling 0% and 100% methylated DNA in the proportions needed to produce the final methylated control.
Sequencing was performed on either a MiSeq or Ion Torrent sequencer. MiSeq runs used the MiSeq Reagent Kit v2 (300 cycles; PN MS-102-2002). PGM runs used either the OT2 200 bp and 200 bp Ion PGM Sequencing Kit (Life Technologies) with a 314 chip or the OT2 400 bp kit and the 400 bp Ion PGM Sequencing Kit with a 314v2 chip. MiSeq sequencing utilized custom sequencing adaptors, as described in the Fluidigm Access Array manual.

All organisms used in this study (listed in S1 File) are maintained at United States Army Medical Research Institute of Infectious Diseases (USAMRIID) or were provided by the Unified Culture Collection (UCC) or the American Type Culture Collection (ATCC, Manassas, VA). Samples included bacterial, parasite DNA, cell culture supernatant from virus-infected cells treated with TRIzol LS (ThermoFisher Scientific, Waltham, MA) or gamma irradiation. Total nucleic acid from each unpurified sample was extracted using the EZ1 Virus Mini Kit v2.0 (Qiagen, Valencia, CA) with the EZ1 robot (Qiagen) according to the manufacturer’s instructions. Total nucleic acid was eluted in 90 μl elution buffer.
Due to a limited supply, Coxiella burnetii DNA was amplified using the REPLI-g Whole Genome Amplification Kit (Qiagen) according to the manufacturer’s instructions. The number of C. burnetii genome equivalents (GE) was approximated using the genome of C. burnetii RSA493 (GenBank# NC_002971) and the C+G (42.7%) and A+T (57.3%) genome percentages. Based on these calculations, 1 GE is approximately 2.05 fg. The approximate number of GE for Plasmodium falciparum 3D7 DNA (ATCC) was similarly determined to be approximately 23.89 fg.

CpGenome human methylated and non-methylated DNA standard set Jurkat DNA (100% methylated) was purchased from Merck (Sydney, NSW, Australia). Whole-genome amplification (WGA) DNA was generated using the protocol of REPLI-g whole genome amplification kit (Qiagen Melbourne, VIC, Australia).