Abstract 437: Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel
Authors: Unselt Desiree , Knudsen Katherine , Rounds Christopher , Doolittle-Hall Janet , Torres Fernando , Sims Jennifer , Mason Jennifer 
Published: June 2022

Abstract
Background: The rapid spread of COVID-19 has resulted in an urgent need for effective diagnostic and therapeutic strategies against SARS-CoV-2. Next-generation sequencing (NGS) is a powerful tool in the identification and characterization of this pathogen and genomic information may aid in understanding the mechanisms of therapeutic resistance, vaccine escape, virulence, and pathogenicity. The Ion AmpliSeq SARS-CoV-2 Research Panel is a targeted NGS solution that facilitates sequence analysis of the SARS-CoV-2 genome. Paired with a bioinformatics assembly and variant calling pipelines, this assay allows for accurate characterization of the dominant SARS-CoV-2 variant. This assay’s performance was analytically validated for the detection of mutations (substitutions, insertions, and deletions) in RNA derived from nasopharyngeal (NP) swabs.
Method: The Ion AmpliSeq SARS-CoV-2 Research panel consists of two primer pair pools generating 237 amplicons specific to the SARS-CoV-2 virus. Reverse transcription of the RNA was performed using the SuperScript VILO cDNA Synthesis kit. Library preparation was then completed using the Ion AmpliSeq Library Kit Plus kit. The final library was quantified, normalized, pooled, and sequenced. Raw sequencing data was aligned to the AmpliSeq SARS-CoV-2 Research panel, using the MN908947.3 reference genome. Variants were called using the Torrent Variant Caller and annotated using the COVID19AnnotateSnpEff plugin. The reference-guided iterative assembler IRMA was used to produce a single consensus sequence consisting of the reference genome sequence modified to include sequence variations supported by the reads. The Pangolin COVID-19 lineage assigner software tool was used to assign SARS-CoV-2 lineage. Analytical validation was completed using controls (Twist Biosciences, BEI Resources, ATCC) and RNA derived from NP swabs. Accuracy and specificity were examined by evaluating the correctness of calling true negative variants compared to false positive and all other variant calls, respectively. Precision and limit of detection (LoD) were examined by evaluating the concordance of variants across replicate samples. Limit of Blank (LoB) was calculated as the 95th percentile of reads per amplicon in the negative samples.
Results: Accuracy of base calling, specificity, and precision were 100% for SNVs, insertions, and deletions above 25% allele frequency. LoD was determined to be 576 viral copies/mL. LoB was determined to be 202 reads per amplicon. Pangolin lineage assignment was 100% for all samples.
Conclusions: This panel accurately characterizes SARS-CoV-2 variants, allowing for accurate consensus sequence generation, mutation annotation, and lineage assignment.
Citation Format: Desiree Unselt, Katherine Knudsen, Christopher Rounds, Janet Doolittle-Hall, Fernando Torres, Jennifer Sims, Jennifer Mason. Characterization of SARS-CoV-2 using the Ion AmpliSeq SARS-CoV-2 research panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 437.