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Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples.

ABSTRACT: Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples.In order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs.The workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.

SUBMITTER: Lewandowska DW

PROVIDER: S-EPMC5549297 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples.

Lewandowska Dagmara W DW Zagordi Osvaldo O Geissberger Fabienne-Desirée FD Kufner Verena V Schmutz Stefan S Böni Jürg J Metzner Karin J KJ Trkola Alexandra A Huber Michael M

Microbiome 20170808 1

<h4>Background</h4>Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnosti ...[more]

PMID: 28789678

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Project description:Bordetella pertussis, the aetiological agent of whooping cough, is re-emerging globally despite widespread vaccination. B. pertussis is highly infectious and, prior to vaccination programmes, was the leading cause of infant mortality. The WHO estimated that over 600?000 deaths are prevented annually by pertussis vaccination, but B. pertussis infection was still responsible for over 63?000 deaths globally in 2013. The re-emergence of B. pertussis has been linked to strains with inactive or absent major virulence factors included in vaccines such as pertactin, pertussis toxin and filamentous haemagglutinin. Thus, the molecular surveillance of currently circulating strains is critical in understanding and controlling B. pertussis. Such information provides data on strains to inform control measures and the identification of future vaccine antigens. Current surveillance and typing methods for B. pertussis rely on the availability of clinical isolates. However, since the 1990s, the majority of pertussis cases have been diagnosed by PCR, where an isolate is not needed. The rapid decline in the availability of B. pertussis isolates impacts our ability to monitor this infection. The growing uptake of next-generation sequencing (NGS) has offered the opportunity for culture-independent genome sequencing and typing of this fastidious pathogen. Therefore, the objective of the study was to optimize respiratory sample preparation, independent of culture, in order to type B. pertussis using NGS. The study compared commercial depletion kits and specimen-processing methods using selective lysis detergents. The goal was to deplete human DNA, the major obstacle for sequencing a pathogen directly from a clinical sample. Samples spiked with a clinically relevant amount of B. pertussis were used to provide comparison between the different methods. Commercial depletion kits including the MolYsis, Qiagen Microbiome and NEBNext Kits were tested. Previously published methods, for Saponin and TritonX-100, were also trialled as a depletion. The ratio of B. pertussis to human DNA was determined by real-time PCR for ERV3 and IS481 (as markers of human and B. pertussis DNA, respectively), then samples were sequenced using the Illumina NextSeq 500 platform. The number of human and B. pertussis sequenced reads were then compared between treatments. The results showed that commercial kits reduced the human DNA present, but also reduced the concentration of target B. pertussis. However, selective lysis with Saponin treatment resulted in almost undetectable levels of human DNA, with minimal loss of target B. pertussis DNA. Sequencing read depth improved five-fold in reads to B. pertussis. Our investigation delivered a potential protocol that will enable the public health laboratory surveillance of B. pertussis in the era of culture-independent testing.

Dataset Information

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples.

Publications

Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples.

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