Ion 540 chip

RNA sequencing was carried out on the indicated samples using a targeted AmpliSeq Transcriptome panel on Ion S5 XL System (ThermoFisher Scientific, Cambridge, MA, USA). In brief, ~30 ng of Turbo DNase treated RNA was used for cDNA synthesis using a SuperScript VILO cDNA Synthesis kit (ThermoFisher Scientific) followed by amplification using Ion AmpliSeq gene expression core panel primers. The enzymatic shearing was performed using FuPa reagent to obtain amplicons of ~200 bp and the sheared amplicons were ligated with the adapter and the unique barcodes. The prepared library was purified using Agencourt AMPure XP Beads (Beckman Coulter, Indianapolis, IN, USA) and the purified library was quantified using an Ion Library TaqMan™ Quantitation Kit (Applied Biosystems, Waltham, MA, USA). The libraries were further diluted to 100 pM and pooled equally with four individual samples per pool. The pooled libraries were amplified using emulsion PCR on Ion OneTouch™ 2 instrument (OT2) and the enrichment was performed on Ion OneTouch™ ES following the manufacturer’s instructions. Thus, prepared template libraries were then sequenced with Ion S5 XL Semiconductor sequencer (ThermoFisher Scientific, Cambridge, MA, USA) using the Ion 540™ Chip.

The Ion AmpliSeq SARS-CoV-2 research panel supplied by Thermo Fisher Scientific contained 247 primer pairs designed to cover the SARS-CoV-2 genome with 125 to 275 bp overlapping amplicons. For cDNA synthesis, the SuperScript VILO cDNA Synthesis Kit (11,754,050, Thermo Fisher Scientific, The Netherlands) was used according to manufacturer’s instructions using 7 μl of diluted nucleic acid solution to an estimated input of 100 copies/reaction using nuclease free water (AM9939, Ambion, Thermo Fisher Scientific, The Netherlands). SARS-CoV-2 whole genome amplification, adapter ligation, and purification were performed using the Ion AmpliSeq SARS-CoV-2 Insight Research Assay (A51305, Thermo Fisher Scientific, The Netherlands) according to manufacturer’s instruction. Libraries were quantified using the Ion Library TaqMan Quantitation Kit (4,468,802, Thermo Fisher Scientific, The Netherlands) according to manufacturer’s instructions. Samples were then sequenced on an Ion GeneStudio S5 system (Thermo Fisher Scientific, The Netherlands) using an Ion 540 chip (Thermo Fisher Scientific, The Netherlands), obtaining approximately up to 1 million paired-end reads per sample.