Ion ampliseq sars cov 2 research panel

The Beta (N501Y, E484K, and K417N) and Delta (L452R and P681R) variants were genotyped using a technique described by Johnson et al. (2022) (link). Briefly, 2.5 μL of RNA with a viral load >133.3GC/cm² was added to 2.5 μL of TaqPath one-step RT-qPCR master mix and 0.25 μL of TaqMan™ SARS-CoV-2 Mutation Panel to make a final volume of 10 μL. The QuantStudio™ 7 Flex Real-Time PCR System instrument (ThermoFisher Scientific, Massachusetts, United States) was used. All RT-qPCR experiments were performed in duplicates, including a non-template control (NTC) and wild-type AcroMetrix COVID-19 RNA control (RUO) (ThermoFisher Scientific) was supplied with the assay with each plate reaction. RNA sequencing was performed at the Central Analytical Facility of Stellenbosch University using the Ion Torrent next-generation sequencing (NGS) technology (ThermoFisher Scientific), with the Ion AmpliSeq SARS-CoV-2 research panel (Thermo Fisher Scientific) according to the manufacturer's instructions.

Standard contact tracing was performed for every SARS-CoV-2-positive HCW (or patient) by the Infection Control Department. To investigate the risk of transmission of HCWs with a positive repeat PCR, potential secondary infections were identified using data from the Occupational Health and Infection Control Department. Potential secondary infections were defined as contacts within the same department that tested positive for SARS-CoV-2 within 7 days after study participants with a repeat RT-PCR Ct value <30 returned to work.
Viral genomes of specimens of study participants and return-to-work contacts were amplified using the Ion AmpliSeq SARS-CoV-2 Research Panel, and sequenced on an Ion GeneStudio S5 system (both from ThermoFisher Scientific, Waltham, MA, USA). Sequences were analysed phylogenetically to infer relatedness in a background of contemporaneous SARS-CoV-2 viral genomes from the Netherlands, derived from the GISAID database (Table S1, see online supplementary material). A maximum-likelihood phylogeny was constructed using the Augur pipeline [17 (link)]. This study used procedures taken from [github.com/nextstrain/ncov] including the clock rate, reference genome and site masking. Trees were visualized using ggtree [18 ] as implemented in R (R Core Team, Vienna, Austria).

Following viral RNA extraction, the DNA library was prepared for complete sequencing using the Ion AmpliSeq SARS-CoV-2 Research Panel (ThermoFisher Scientific, USA) according to the manufacturer's instructions. This panel describes a targeted amplicon sequencing approach that includes 237 amplicons specific to SARS-CoV-2 with amplicon sizes ranging from 125 to 275 bp. Preparing the library included the following steps: 1) RNA reverse transcription using the ThermoFisher Scientific SuperSrcriptTM VILO cDNA Synthesis Kit (ThermoFisher Scientific, USA), 2) target amplification for 15–20 cycles, 3) partial amplicon digestion, 4) adapter ligation 5) library purification using Agencourt™ AMPure™ XP reagent at room temperature, and 6) quantification of the amplified library by Qubit™ Fluorometer kit. Barcoded libraries were loaded to the Ion Chef™ Instrument (Thermo Fisher Scientific) for emulsion PCR, enrichment, and loading onto the Ion Chip followed by high-throughput sequencing reactions run on Ion GeneStudio S5, ion torrent sequencer (ThermoFisher Scientific).