RT Journal Article
SR Electronic
T1 Metagenomic Sequencing for Combined Detection of RNA and DNA Viruses in Respiratory Samples from Paediatric Patients
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 492835
DO 10.1101/492835
A1 Sander van Boheemen
A1 Anneloes L. van Rijn-Klink
A1 Nikos Pappas
A1 Ellen C. Carbo
A1 Ruben H.P. Vorderman
A1 Peter J. van `t Hof
A1 Hailiang Mei
A1 Eric C.J. Claas
A1 Aloys C.M. Kroes
A1 Jutte J.C. de Vries
YR 2018
UL http://biorxiv.org/content/early/2018/12/10/492835.abstract
AB Introduction Viruses are the main cause of respiratory tract infections. Metagenomic next-generation sequencing (mNGS) enables the unbiased detection of all potential pathogens in a clinical sample, including variants and even unknown pathogens. To apply mNGS in viral diagnostics, there is a need for sensitive and simultaneous detection of RNA and DNA viruses. In this study, the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections, with single tube DNA and RNA sample-pre-treatment and potential for automated pan-pathogen detection was studied.Materials and Methods The sequencing protocol and bioinformatics analysis was designed and optimized including the optimal concentration of the spike-in internal controls equine arteritis virus (EAV) and phocine-herpes virus-1 (PhHV-1).The whole genome of PhHV-1 was sequenced and added to the NCBI database. Subsequently, the protocol was retrospectively validated using a selection of 25 respiratory samples with in total 29 positive and 346 negative PCR results, previously sent to the lab for routine diagnostics.Results The results demonstrated that our protocol using Illumina Nextseq 500 sequencing with 10 million reads showed high repeatability. The NCBI RefSeq database as opposed to the NCBI nucleotide database led to enhanced specificity of virus classification. A correlation was established between read counts and PCR cycle threshold value, demonstrating the semi-quantitative nature of viral detection by mNGS. The results as obtained by mNGS appeared condordant with PCR based diagnostics in 25 out of the 29 (86%) respiratory viruses positive by PCR and in 315 of 346 (91%) PCR-negative results. Viral pathogens only detected by mNGS, not present in the routine diagnostic workflow were influenza C, KI polyomavirus, and cytomegalovirus.Conclusions Sensitivity and analytical specificity of this mNGS protocol was comparable with PCR and higher when considering off-PCR target viral pathogens. All potential viral pathogens were detected in one single test, while it simultaneously obtained detailed information on detected viruses.