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. Clinical microbiology is one of the most rapidly changing areas of laboratory medicine today due to the introduction of new technologies and automation (1). Molecular testing, such as PCR, is becoming the de facto gold standard for the detection of pathogens that are difficult to culture by offering high sensitivity and specificity and a rapid turnaround time (2). Syndrome-based multiplex molecular assays can detect up to 30 of the most common pathogens associated with respiratory infections, gastroenteritis, and central nervous system (CNS) infections (3-6). However, the complete list of infectious agents associated with these infections greatly exceeds the capabilities of even the best multiplex assays. These less common organisms, for which tests are not readily available, are likely responsible for many cases of undiagnosed illness, particularly in critically ill patients and those with compromised immunity (7,8). Therefore, there is increasing interest in the application of novel technologies, such as next-generation sequencing (NGS), for unbiased detection of pathogens in clinical samples.Among the various challenges with the implementation of NGS for routine pathogen detection using metagenomics, the presence of an overwhelming amount of host DNA is one of the most important problems to be addressed. A previous metagenomic study on nasopharyngeal aspirate samples from patients with acute lower respiratory tract infections revealed that up to ϳ95% of raw NGS reads were of human DNA (9). The su...

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing

“…Additionally, previous studies using the MolYsis kit focused on improving techniques such as real-time PCR, whereas use of these methods for WGS, where microbial DNA enrichment could be even more useful, has been less studied (Votintseva et al, 2015). Other methods such as host cell lysis with detergents (Hasan et al, 2016) or ox bile (Zhou & Pollard, 2012) and immunoprecipitation of DNA with inactive methyl-specific restriction endonucleases (Barnes et al, 2014; Liu et al, 2016) have also been reported, but are not available as commercial products.…”

Metagenomic sequencing with spiked primer enrichment for viral diagnostics and genomic surveillance

“…An alternative approach for pathogen enrichment in mNGS libraries is the use of a host depletion method. Such methods include differential lysis 37 , the removal of abundant human ribosomal and mitochondrial RNA sequences using antibody hybridization or depletion of abundant sequences by hybridization (DASH) 38 and nuclease treatment before extraction 39,40 . Aside from a post-extraction DNase step, we did not incorporate host depletion as part of MSSPE, given that these methods can be cumbersome, with additional Abbott m2000 RT-PCR assays were used to estimate the titres of HIV and HCV; viral titres for other viruses were estimated using in-house qRT-PCR assays with standard curve analysis.…”

“…d Absolute percentage increase from using random primer only (coverage by SP (%) − coverage by RH (%)); coverage of 40-60% is sufficient for genotypic and phylogenetic inference from partial genome assemblies 27 steps, and difficult to standardize. Furthermore, host depletion methods can bias detection towards specific pathogen types, such as the enrichment of bacteria and fungi with cell walls with differential lysis 37 , or encapsidated viruses with pre-extraction nuclease treatment 39,40 . Host depletion can also be performed at the RNA level by the use of non-human primers to bias against abundant human host ribosomal and/or mitochondrial RNA 41,42 .…”

Retrospective Validation of a Metagenomic Sequencing Protocol for Combined Detection of RNA and DNA Viruses Using Respiratory Samples from Pediatric Patients

“…Therefore, this study has initiated the optimization of several steps throughout the presequencing and postsequencing workflow, which are considered essential for sensitive and specific mNGS-based virus detection. Many virus discovery or virus diagnostic protocols have focused on the enrichment of viral particles 32 with the intention to increase the relative amount of virus reads. However, these methods are laborious and intrinsically exclude viral nucleic acid located in host cells.…”