Ion torrent genexus integrated sequencer

For the whole viral genome sequencing, a volume of 25 µl of a tenfold dilution of each sample was distributed in an Applied Biosystems™ MicroAmp™ EnduraPlate™ Optical 96-Well Clear Reaction Plate. The plate was sealed with a sheet of adhesive PCR plate foil and was placed on the Ion Torrent Genexus Integrated Sequencer (ThermoFisher Scientific, San Diego, CA).
All the following steps, including cDNA synthesis, library preparation and equalization, template preparation, sequencing and post-run analysis were fully automated on the Genexus system.
The samples were sequenced on two Ion Torrent™ GX5™ Chip. The sequencing run was performed by using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer (ThermoFisher Scientific, San Diego, CA), according to two preinstalled assays: “SARS-CoV-2 Low Titer Research Assay” for samples with viral load < 200 copies and “SARS-CoV-2 Research Assay” for > 200 copies.
The Ion AmpliSeq SARS-CoV-2 Research Panel consists of 2 primer pools targeting 237 amplicons tiled across the SARS-CoV-2 genome, with an additional 5 primer pairs targeting human expression controls. The SARS-CoV-2 amplicons range from 125 to 275 bp in length.
Post-sequencing run analysis was performed by Genexus Software with following plug-in: COVID19AnnotateSnpEff for variant annotation, IRMA for genome-assisted and Trinity for de novo sequence assembly.

The nucleic acid extraction was conducted using either the Maxwell RSC Instrument (Promega, Madison, WI, USA) in combination with the Maxwell RSC FFPE Plus DNA kit or the Maxwell RSC RNA FFPE kit (Promega). After the extraction of nucleic acid, the concentration was measured by employing the Qubit Fluorometric quantification assay (Thermo Fisher Scientific, Waltham, MA, USA), utilizing the Qubit RNA HS Assay Kit and the Qubit dsDNA HS Assay Kit. The Ion Torrent™ Genexus™ Integrated Sequencer (Thermo Fisher Scientific) was used for the detection of genomic alterations by Ion semiconductor sequencing (Ion Torrent™ Technology, Thermo Fisher Scientific). The Oncomine™ Precision Assay GX panel (OPA) provided by Thermo Fisher Scientific was utilized, targeting 50 key genes. Among them, 45 genes were designed for DNA mutation detection, 18 for fusion detection, and 14 for copy number variant (CNV) detection. In addition, the panel incorporated a 5′/3′ expression imbalance strategy for the detection of novel fusions. By using this panel, up to 16 samples could be sequenced simultaneously on a single run with the Genexus sequencer.