Whole Genome Amplification of Microbial DNA from Host-Depleted Clinical Samples

Abstract Clinical metagenomics (CMg) involves the untargeted sequencing of the genetic content of samples collected from patients and is a highly promising method for the diagnosis of infectious disease. Depending on the sample type, CMg can be reliant on the removal of the host genetic material from the sample to support detection of microbial pathogens, and this selective process (or an otherwise low abundance of microbial cells in the sample) may result in concentrations of DNA too low for productive sequencing. Whole genome amplification (WGA), the nonselective amplification of the total DNA of a sample, can be applied to significantly increase the concentration of DNA and enable CMg sequencing. This chapter describes the methods for the amplification of microbial DNA extracted from host-depleted wound swab samples using the GenomiPhi ™ V3 Ready-To-Go ™ (Cytiva) DNA WGA kit and host-depleted whole blood samples using the REPLI-g ® Single-Cell WGA kit (Qiagen). This is followed by the de-branching and bead-based clean-up of the amplified DNA, resulting in highly concentrated DNA ready for CMg DNA sequencing.