The respiratory virome and exacerbations in patients with chronic obstructive pulmonary disease

van Rijn, Anneloes L.; van Boheemen, Sander; Sidorov, Igor; Carbo, Ellen C.; Pappas, Nikos; Mei, Hailiang; Feltkamp, Mariet; Aanerud, Marianne; Bakke, Per; Claas, Eric C. J.; Eagan, Tomas M.; Hiemstra, Pieter S.; Kroes, Aloys C. M.; de Vries, Jutte J. C.

The metagenomics protocol used has been described and optimized for simultaneously RNA and DNA detection previously [14] . In short, internal controls, Equine Arteritis virus (EAV) for RNA and Phocid Herpesvirus-1 (PhHV) for DNA (kindly provided by prof. dr. H.G.M. Niesters, the Netherlands), were spiked in 200 μl of the virus transport medium in which the nasopharyngeal swab was stored. Nucleic acids were extracted directly from 200 μl clinical sample using the Magnapure 96 DNA and Viral NA Small volume extraction kit on the Mag-naPure 96 system (Roche Diagnostics, Almere, The Netherlands) with 100 μL output eluate (an updated version of the isolation method used for qPCR, tested previously [20] ). Extraction buffer was used as negative control (for extraction, library preparation, and sequencing). For library preparation, 7 μl of nucleic acids were used, using the NEBNext 1 Ultra™ Directional RNA Library Prep Kit for Illumina 1 , with several in-house adaptations to the manufacturers protocol in order to enable simultaneous detection of both DNA and RNA. The following steps were omitted: poly A mRNA capture isolation, rRNA depletion and DNase treatment step. This resulted in a single tube per sample throughout library preparation containing both DNA and RNA. Metagenomic sequencing was performed on an Illumina NextSeq 500 sequencing system (Illumina, San Diego, CA, USA), and approximately 10 million 150 bp paired-end reads per sample were obtained.