title: Impact of Contaminating DNA in Whole-Genome Amplification Kits Used for Metagenomic Shotgun Sequencing for Infection Diagnosis.
abstract: Whole-genome amplification (WGA) is a useful tool for amplification of very small quantities of DNA for many uses, including metagenomic shotgun sequencing for infection diagnosis. Depending on the application, background DNA from WGA kits can be problematic. Three WGA kits were tested for their utility in a metagenomics approach to identify the pathogens in sonicate fluid comprised of biofilms and other materials dislodged from the surfaces of explanted prosthetic joints using sonication. The Illustra V2 Genomiphi, Illustra single cell Genomiphi, and Qiagen REPLI-g single cell kits were used to test identical sonicate fluid samples. Variations in the number of background reads, the genera identified in the background, and the number of reads from known pathogens known to be present in the samples were observed between kits. These results were then compared to those obtained with a library preparation without prior WGA using an NEBNext Ultra II paired-end kit, which requires a very small amount of input DNA. This approach also resulted in the presence of contaminant bacterial DNA and yielded fewer reads from the known pathogens. These findings highlight the impact that WGA kit selection can have on metagenomic analysis of low-biomass samples and the importance of the careful selection and consideration of the implications of using these tools.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Thoendel
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Patricio
authors.identifier: 0000-0003-0282-4217
authors.initials: P
authors.lastname: Jeraldo
authors.affiliation: Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Kerryl E
authors.initials: KE
authors.lastname: Greenwood-Quaintance
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Yao
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Chia
authors.affiliation: Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Arlen D
authors.initials: AD
authors.lastname: Hanssen
authors.affiliation: Department of Orthopedic Surgery, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Abdel
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA patel.robin@mayo.edu.|Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota, USA.
authors.forename: Robin
authors.identifier: 0000-0001-6344-4141
authors.initials: R
authors.lastname: Patel
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D056186:Metagenomics / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D019736:Prostheses and Implants / Q000382:microbiology*; D016459:Prosthesis-Related Infections / Q000175:diagnosis; D000073336:Whole Genome Sequencing / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
keywords: metagenomics; prosthetic joint infection; whole-genome amplification