Ion s5 sequencing kit

An exome amplicon library was prepared using the Ion AmpliSeq Exome RDY kit (Thermo Fisher, Waltham, MA, USA). Briefly, 100 ng of genomic DNA was added to dehydrated, ultra-high multiplexed primer pairs (12 pools) in a 96-well plate and amplified with the following PCR conditions: 99 °C for 2 min; 99 °C for 15 s and 60 °C for 16 min (10 cycles); and holding at 10 °C. Primers were partially digested using a FuPa reagent, and then sequencing motifs and barcodes were ligated to the amplicons. The library was purified using the Agencourt AMPure XP Reagent (Beckmann Coulter, Brea, CA, USA). The concentration of the final library was determined using the Ion Library TaqMan Quantitation Kit (Thermo Fisher, Waltham, MA, USA) on an ABI 7500 qPCR instrument with the absolute quantification method.
Template preparation was performed with the Ion 540 OT2 Kit (Thermo Fisher, Waltham, MA, USA) on semi-automated Ion OneTouch 2 instrument using the emPCR method. After breaking the emulsion, non-templated beads were removed from the solution during the enrichment process on the Ion OneTouch ES (Thermo Fisher, Waltham, MA, USA) machine. Subsequently, the sequencing primer and polymerase were added, the fully prepared Ion sphere particles were loaded into an Ion 540 chip, and sequencing runs were performed using the Ion S5 Sequencing kit (Thermo Fisher, Waltham, MA, USA) with 500 flows.

The Oncomine™ Tumor Mutation Load Assay is a PCR-based target enrichment next-generation sequencing assay performed on the Ion Torrent platform. The panel covers 1.65 Mb with 1.2 Mb of exonic bases across 409 oncogenes relevant across major cancer types. DNA was extracted from formalin-fixed paraffin-embedded tissue using the RecoverAll Multi-Sample RNA/DNA Workflow kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s protocol. Genomic DNA was quantified by quantitative real-time reverse transcription (RT)-PCR using a TaqMan® RNase P Detection Reagents kit (Applied Biosystems, Foster City, CA, USA). The Libraries were prepared by automation using The Ion AmpliSeq™ Kit for Chef DL8 (Ion Torrent, Thermo Fisher Scientific). Library preparation and templating were performed on the Ion Chef using an automated Ion Ampliseq^TM Kit for Chef DL8 (Ion Torrent) and Ion 540 chip - Chef kit (Ion Torrent) respectively. Sequencing was performed on the Ion S5™XL Sequencer using an Ion S5™sequencing kit (Thermo Fisher Scientific) with Torrent Suite software (Version 5.10). Variants were identified using the Ion Torrent Variant Caller plug-in (Version 5.10) and annotated using Ion Reporter software (Version 5.10).

Nine K. pneumoniae and five E. coli isolates were submitted for whole-genome sequencing (WGS) using the Ion S5 system, the Ion S5 sequencing kit, and the Ion 530 chip, following the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The genomes were mapped using the Galaxy platform [27 (link)], and the Klebsiella pneumoniae strain MGH78578 (GenBank accession number CP000647.1) and Escherichia coli strain K-12 substr. MG1655 (GenBank accession number NC_000913.3) were used as a reference for molecular characterization. Multi-locus sequence typing (MLST) and resistome analysis were performed using MLSTFinder 2.0 [28 (link)] and ResFinder 4.0 tolls [29 (link)], respectively. The whole-genome shotgun project of K. pneumoniae and E. coli isolates was deposited at the DDBJ/EMBL/GenBank under the Bioproject: PRJNA1100951.