Ion s5xl instrument

Mutation analysis was performed by targeted sequencing using the Ion Torrent platform (Thermo Fisher Scientific, Waltham, MA, USA) and a custom-designed sequencing gene panel (Ion AmpliSeq, Thermo Fisher Scientific) encompassing 525 amplicons covering coding regions of 58 GC related genes. The multiplex PCR based Ion AmpliSeq targeted sequencing technology (Thermo Fisher Scientific) was used for DNA library preparation and amplification of target regions using the Ion AmpliSeq Library Kit v2.0, as well as the specific GC sequencing panel consisting of four primer pools as described in detail previously [19 , 20 (link)]. Automated template preparation of the final libraries as well as chip loading (Ion 520, 530, or 540) was performed on an Ion Chef instrument and sequenced using an Ion S5XL instrument (Thermo Fisher Scientific). Data analysis was performed referring to Pfarr et al. [20 (link)] and ANNOVAR was used to annotate the sequence variants [21 (link)].

Total RNA from an ultracentrifugation pellet was fragmented and reverse-transcribed into cDNA with R6 primers using RevertAid reverse transcriptase (ThermoFisher Scientific, Waltham, MA, USA) followed by second strand synthesis with the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs Inc., Ipswich, MA, USA). Resultant double-stranded DNA was purified using Ampure XP (Beckman Coulter, Brea, CA, USA) and subjected as an input for library preparation process using the NEBNext^® Fast DNA Library Prep Set for Ion Torrent™ (New England Biolabs Inc., Ipswich, MA, USA) following the manufacturer’s instructions. The resultant DNA library was quantified with the Ion Library TaqMan™ Quantitation Kit (ThermoFisher Scientific, Waltham, MA, USA) followed by templating on the Ion Chef instrument (ThermoFisher Scientific, Waltham, MA, USA) and sequencing on the Ion S5XL instrument, with the viral library constituting a part of the Ion 530 Chip. Raw reads were quality-controlled using FaQCs v2.09 [25 (link)] and assembled into contigs using SPAdes v3.13.0 [26 (link)] with iontorrent flag. The resultant contigs were screened for viral sequences using Blastn algorithm in BLAST v2.9.0+ with nt database and contigs corresponding to virus were extracted for further investigation.

Total RNA was extracted from the ultracentrifugation pellet using TRI Reagent LS. RNA was fragmented and reverse-transcribed into cDNA with R6 primers using RevertAid reverse transcriptase (ThermoFisher Scientific, Waltham, MA, USA), followed by second-strand synthesis with the NEBNext Ultra II Non-Directional RNA Second-Strand Synthesis Module (New England Biolabs Inc., Ipswich, MA, USA). Resultant double-stranded DNA was purified using Ampure XP (Beckman Coulter, Brea, CA, USA) and subjected as an input for library preparation using the NEBNext^® Fast DNA Library Prep Set for Ion Torrent™ (New England Biolabs Inc., Ipswich, MA, USA) following the manufacturer’s instructions. The resultant DNA library was quantified with the Ion Library TaqMan™ Quantitation Kit (ThermoFisher Scientific, Waltham, MA, USA), followed by templating on the Ion Chef instrument (ThermoFisher Scientific, Waltham, MA, USA) and sequencing on the Ion S5XL instrument with the viral library constituting a part of the Ion 530 Chip. Raw reads were filtered by quality (q20) and length (>35) using Trimmomatic v.0.39 [34 (link)] and mapped on the strain Miass527 genome using the UGENE v1.32.0 “map NGS Reads to Reference” function with 10% of mismatches allowed. After mapping, virus consensus sequence was exported using default UGENE settings [35 (link)].