Abstract 707: Detection of SARS-CoV-2 co-infections with seasonal respiratory viruses using a multiplexed amplicon NGS panel
Authors: Lee Lucie S. , Feigelman Rounak , Debruyne David , Spencer Julia , Li Chenyu , Liu Zhitong , Liu Guoying 
Published: July 2021

Abstract
The ongoing COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected everyone around the world. Most symptoms of COVID-19 overlap with symptoms of the flu and the common cold. With the approaching flu season, early and accurate detection of these viruses are critical not only to slow the spread of SARS-CoV-2, but also to ensure that patients receive the appropriate care based on their diagnosis, and to keep the healthcare system from becoming overloaded. We designed a next-generation sequencing (NGS) assay to detect the SARS-CoV-2, influenza A H1N1, influenza A H3N2, influenza B, RSV A, and RSV B viruses, and to allow for surveillance and subtype identification. The primers for this multiplex PCR-based method interrogate nearly every gene of each virus. Subsequent sequencing enables not only virus detection but also co-infection detection for these respiratory viruses. In addition, sequencing of virus-positive samples makes it possible to use this assay for mutation and strain analysis. Using the CleanPlex target enrichment technology, we prepared targeted amplicon libraries with human total RNA (50ng) spiked with SARS-CoV-2 synthetic RNA, influenza A synthetic RNA, influenza B synthetic RNA, RSV A RNA, and RSV B RNA (100-10,000 viral genome copies), either individually or in combination. The NGS library preparation protocol includes a reverse transcription step to generate cDNA, followed by a multiplexed PCR step for specific amplification of viral targets. A critical subsequent background cleaning step removes non-specific PCR products, and a final PCR adds sample indices and Illumina sequencing adapters to complete the NGS library preparation. Libraries were sequenced on the Illumina iSeq100 platform, and sequencing metrics as well as virus characterization were determined using Paragon Genomics's bioinformatics pipeline. Each sample was sequenced at 1,000 average paired-end reads per amplicon. After aligning sequencing reads to each viral genome, we were able to detect nearly every gene of each virus with high specificity. At viral loads of 10,000 copies, 96-100% of amplicons were covered at &gt;100X, and mapped to the intended virus species as well as to the intended subtypes for influenza A and RSV. The assay also accurately identified co-infected samples, which contained equal copies of SARS-CoV-2 and influenza (either A H1N1, A H3N2, or B). In summary, the assay detects multi-target respiratory viruses with high accuracy and specificity. We plan to expand on our findings for this assay's performance with additional targets, co-infection studies and mutational analysis studies.
Citation Format: Lucie S. Lee, Rounak Feigelman, David Debruyne, Julia Spencer, Chenyu Li, Zhitong Liu, Guoying Liu. Detection of SARS-CoV-2 co-infections with seasonal respiratory viruses using a multiplexed amplicon NGS panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 707.