Rapid metagenomic characterization of a case of imported COVID-19 in Cambodia

Jessica E. Manning; Jennifer A. Bohl; Sreyngim Lay; Sophana Chea; Ly Sovann; Yi Sengdoeurn; Seng Heng; Chan Vuthy; Katrina Kalantar; Vida Ahyong; Michelle Tan; Jonathan Sheu; Cristina M. Tato; Joseph L. DeRisi; Laurence Baril; Veasna Duong; Philippe Dussart; Erik A. Karlsson

Nasopharyngeal and oropharyngeal swabs (combined into one tube) were collected from a symptomatic patient meeting case definition for possible infection with COVID-19 pneumonia on January 26, 2020. Extraction of viral nucleic acids from clinical sample was performed with a QIAamp Viral RNA Mini Kit (Qiagen #52906) as described by manufacturer. Extracted RNA samples were DNAse-treated and tested via real-time polymerase chain reaction for SARS-CoV-2 using both the Charité Virology and Hong Kong University protocols and confirmed positive on January 27 th , 2020. 7 mNGS preparation and analysis On January 29, samples were received for cDNA library preparation. Since clinical specimens are obtained from suspected COVID-19 cases as part of the national outbreak response, requirement for informed consent was waived for pathogen identification and characterization. mNGS libraries were prepared from the viral RNA extracted from the sample. cDNA was converted to Illumina libraries using the NEBNext Ultra II DNA Library Prep Kit (E7645) according to the manufacturer's recommendations. Library size and concentration were determined using the 4150 Tapestation system (Agilent, MA, USA). External RNA Controls 103 Consortium collection (ThermoFisher, 4456740) spike-in controls were used as markers of potential library preparation errors and for input RNA mass calculation. Sample was sequenced on an Illumina iSeq100 instrument using 150 nucleotide paired-end sequencing. A water ("notemplate") control was included in library preparation. Microbial sequences from the sample are located in the National Center for Biotechnology Information (NCBI) Sequence Read Archive.