Incubation periods and transmissibility of COVID-19 are questions of current worldwide concern, but difficult to answer given nonspecific symptoms that represent a variety of respiratory viruses. In this setting, genetic characterization of the virus from geographically diverse patient samples is key to infer the rate of spread. However, as the virus reaches resource-limited settings such as Cambodia, Laos, and Myanmar in close proximity to the outbreak’s epicenter, there are basic challenges in sample collection, contact tracing, and surveillance that hinder disease containment, much less the ability to sequence new cases in-country. Implementing in-country sequencing and post-sequencing data analysis speeds up the time to pathogen identification, giving scientists in-country the ability to inform leading public health officials to combat emerging infections.
In a rapidly implemented response to the nCOV-2019 outbreak (manuscript), the NIH-CNM team and the Institut Pasteur du Cambodge used metagenomic next-generation sequencing (mNGS) and the IDseq bioinformatics platform to review the Cambodian index nCOV-2019 case in less than 48 hours from sample receipt.
This project had 2 goals:
The Institut Pasteur du Cambodge, National Referral Laboratory for COVID-19, performed the RNA extraction from a nasopharyngeal swab from the index case. On February 1st, the NIH-CNM team made sequencing libraries from the extracted RNA, sequenced the samples on an iSeq100, demultiplexed the FASTQ files, and the results of the sequencing run were compiled into Illumina’s Basespace. The data was then uploaded to IDseq and processed using the latest IDseq database - updated from NCBI on 2019-09-17. While the reference database did not contain the reference sequences for SARS-CoV-2, which were deposited to NCBI in January 2020, conclusions could still be drawn from the data. IDseq’s NCBI reference database was updated on 2019-02-10 with the most recent version of NCBI that included the recently added SARS-CoV-2 sequences. The analysis was rerun using the new NCBI reference database to confirm the match to SARS-CoV-2 in the PCR-positive index case. Further experiments were done using an enrichment protocol to isolate a full genome sequence for SARS-CoV-2 from the Index Patient. The sequence generated during the current study was submitted to the GISAID repository (GISAID EPI ISL 411902), and is available for comparison with other SARS-CoV-2 sequences, globally, on Nextstrain.