title: Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing.
abstract: Next-generation sequencing (NGS) technology has shown promise for the detection of human pathogens from clinical samples. However, one of the major obstacles to the use of NGS in diagnostic microbiology is the low ratio of pathogen DNA to human DNA in most clinical specimens. In this study, we aimed to develop a specimen-processing protocol to remove human DNA and enrich specimens for bacterial and viral DNA for shotgun metagenomic sequencing. Cerebrospinal fluid (CSF) and nasopharyngeal aspirate (NPA) specimens, spiked with control bacterial and viral pathogens, were processed using either a commercially available kit (MolYsis) or various detergents followed by DNase prior to the extraction of DNA. Relative quantities of human DNA and pathogen DNA were determined by real-time PCR. The MolYsis kit did not improve the pathogen-to-human DNA ratio, but significant reductions (>95%;P< 0.001) in human DNA with minimal effect on pathogen DNA were achieved in samples that were treated with 0.025% saponin, a nonionic surfactant. Specimen preprocessing significantly decreased NGS reads mapped to the human genome (P< 0.05) and improved the sensitivity of pathogen detection (P< 0.01), with a 20- to 650-fold increase in the ratio of microbial reads to human reads. Preprocessing also permitted the detection of pathogens that were undetectable in the unprocessed samples. Our results demonstrate a simple method for the reduction of background human DNA for metagenomic detection for a broad range of pathogens in clinical samples.
authors.affiliation: Department of Pathology, Sidra Medical and Research Center, Doha, Qatar Weill Cornell Medical College in Qatar, Doha, Qatar mhasan@sidra.org.
authors.forename: Mohammad R
authors.identifier: http://orcid.org/0000-0002-4658-7949
authors.initials: MR
authors.lastname: Hasan
authors.affiliation: Division of Biomedical Informatics Research, Sidra Medical and Research Center, Doha Qatar.
authors.forename: Arun
authors.initials: A
authors.lastname: Rawat
authors.affiliation: Department of Pathology, Sidra Medical and Research Center, Doha, Qatar.
authors.forename: Patrick
authors.initials: P
authors.lastname: Tang
authors.affiliation: Division of Biomedical Informatics Research, Sidra Medical and Research Center, Doha Qatar.
authors.forename: Puthen V
authors.initials: PV
authors.lastname: Jithesh
authors.affiliation: Department of Pathology, Sidra Medical and Research Center, Doha, Qatar.
authors.forename: Eva
authors.initials: E
authors.lastname: Thomas
authors.affiliation: Department of Pathology, Sidra Medical and Research Center, Doha, Qatar Children's and Women's Health Centre of BC, PHSA, Vancouver, British Columbia, Canada.
authors.forename: Rusung
authors.initials: R
authors.lastname: Tan
authors.affiliation: Children's and Women's Health Centre of BC, PHSA, Vancouver, British Columbia, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Peter
authors.initials: P
authors.lastname: Tilley
mesh_terms: D002555:Cerebrospinal Fluid / Q000382:microbiology / Q000821:virology; D003141:Communicable Diseases / Q000175:diagnosis*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009305:Nasopharynx / Q000382:microbiology / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*