Demultiplexed raw sequencing data files (fastq.gz

Böni, Jürg

Geissberger, Fabienne-Desirée

Huber, Michael

Kufner, Verena

Lewandowska, Dagmara W

Metzner, Karin J

Schmutz, Stefan

Trkola, Alexandra

Zagordi, Osvaldo

Microbiome

Abstract Background 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. Results 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. Conclusions 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

Dagmara W. Lewandowska

Osvaldo Zagordi

Fabienne-Desirée Geissberger

Verena Kufner

Stefan Schmutz

Jürg Böni

Karin J. Metzner

Alexandra Trkola

Michael Huber

Directory of Open Access Journals

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

NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY

Crossref

ZENODO

ZORA

http://www.zora.uzh.ch/id/eprint/140275/1/s40168-017-0317-z.pdf

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

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals

NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY

Crossref

ZENODO

NEUROSURGERY ENTHUSIASTIC WOMEN SOCIETY

ZORA