Ion s5 sequencing reagents

We performed target deep sequencing of the genomic DNA extracted from the three CRC tissue samples and matched normal samples using a custom NGS panel (OncoChase-AS01, ConnectaGen, Seoul, Korea), targeting 95 cancer-related genes as described elsewhere [10 (link),11 (link)]. Tumor DNA was amplified, digested, and barcoded using the Ion Ampliseq library kit 2.0 (Thermo Fisher Scientific) and Ion Xpress barcode adapter kit (Thermo Fisher Scientific) as described elsewhere [10 (link)]. The libraries were then templated on an Ion Chef system (Thermo Fisher Scientific) using Ion 520 and Ion 530 Chef reagents (Thermo Fisher Scientific). The prepared libraries were sequenced on an Ion S5 sequencer using an Ion 530 chip and Ion S5 sequencing reagents (Thermo Fisher Scientific) as described elsewhere [10 (link)].

For whole viral genome sequencing, total RNA was reverse transcribed using Invitrogen SuperScript VILO^TM cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). One hundred and seven samples were analyzed in eight different sequencing runs using the Ion Torrent S5 system (Thermo Fisher Scientific, Waltham, MA, USA) after library preparation, consisting of fragmentation and adapter ligation onto the PCR products and clonal amplification. cDNA libraries were then prepared using the Ion AmpliSeq SARS-CoV-2 Research Panel (Thermo Fisher Scientific, Waltham, MA, USA). After quantification of cDNA libraries with Real-Time Step One PCR System (Thermo Fisher Scientific, Waltham, MA, USA), the prepared samples of ion sphere particles (ISP) were loaded onto an Ion 520™ chip with the Ion Chef (Thermo Fisher Scientific, Waltham, MA, USA). Sequencing was performed using the Ion S5™ sequencing reagents (Thermo Fisher Scientific, Waltham, MA, USA). The Torrent Suite 5.14.0 platform and specific plugins were used for NGS data analysis. All analysed sequences showed an alignment accuracy of over 96% and a base coverage over 20× (Figure 1). The pangolin software was used for the assignment of SARS-CoV-2 lineages. All sequences were then submitted as FASTA files on gisaid.org, which provides open access to genomic data on SARS-CoV-2.