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Depletion of Human DNA in Spiked Clinical Specimens for Improvement of Sensitivity of Pathogen Detection by Next-Generation Sequencing
- Source :
- Journal of clinical microbiology. 54(4)
- Publication Year :
- 2015
-
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.
- Subjects :
- 0301 basic medicine
Microbiology (medical)
030106 microbiology
Human pathogen
Shotgun
Biology
Real-Time Polymerase Chain Reaction
Communicable Diseases
Sensitivity and Specificity
DNA sequencing
Specimen Handling
03 medical and health sciences
chemistry.chemical_compound
Nasopharyngeal aspirate
Nasopharynx
Humans
Pathogen
Cerebrospinal Fluid
High-Throughput Nucleotide Sequencing
Bacteriology
Molecular biology
030104 developmental biology
chemistry
Molecular Diagnostic Techniques
Metagenomics
Human genome
DNA
Subjects
Details
- ISSN :
- 1098660X
- Volume :
- 54
- Issue :
- 4
- Database :
- OpenAIRE
- Journal :
- Journal of clinical microbiology
- Accession number :
- edsair.doi.dedup.....c85d8c19ae3cb10b0396147ed86acd47