Ion dna barcoding kit

An Ion Torrent adapter‐ligated library was generated following the manufacturer’s protocol (Ion AmpliSeq™ Exome RDY kit PIv3, Rev. A.0; MAN0010084; Thermo Fisher Scientific, Inc.). Briefly, 100 ng high‐quality genomic DNA was used to prepare the Ion AmpliSeq™ Exome capture library. Pooled amplicons were end‐repaired, and Ion Torrent adapters and amplicons were ligated with DNA ligase. Following AMPure bead purification (Beckman Coulter, Inc), the library’s concentration and size were determined using the Applied Biosystems^® StepOne™ Real‐Time PCR system and Ion Library TaqMan^® Quantitation kit (both from Thermo Fisher Scientific, Inc.).
Sample emulsion PCR, emulsion breaking, and enrichment were performed using the Ion PI™ Hi‐Q™ Chef 200 kit (Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. An input concentration of one DNA template copy per ion sphere particles (ISPs) was added to the emulsion PCR master mix and the emulsion was generated using the Ion Chef™ System (Thermo Fisher Scientific, Inc.). Template‐positive ISPs were enriched, sequencing was performed using Ion PI™ Chip kit v3 chips on the Ion Torrent Proton, and barcoding was performed using the Ion DNA Barcoding kit (Thermo Fisher Scientific, Inc.).

A subset of the clinical isolates was analyzed using whole-genome sequencing (WGS). The bacterial genomes were sequenced using the IonTorrent PGM platform (Life Technologies, Carlsbad, USA) in accordance with the manufacturer’s instructions. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies), and its size was selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analyzed using the High Sensitivity kit (Agilent, Santa Clara, USA). Further sample processing was performed using the Ion OneTouch kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). The single reads obtained were de novo assembled using MIRA 3.9.9, which is a part of the Assembler plugin on the Ion Torrent server. The contigs were analyzed using the ResFinder version 4.1. web-service [80 (link)].

Isolates were transported to the Molecular Diagnostics group at the Austrian Institute of Technology where they were checked for viability at arrival. Fifteen isolates were not viable after transportation, and sequencing failed in three isolates. After a viability check of all isolates, genomic DNA was extracted with the QiAmp DNA mini kit (Qiagen, Hilden, Germany). Whole genome sequencing (WGS) was performed using the Ion Torrent PGM platform using 400 bp read chemistry. Sequencing was performed according to the protocol recommended by Life Technologies. The Ion Xpress Plus Fragment Library Kit was used to enzymatically shear 100 ng of the genomic DNA. The target fragment size was 400 bp. Subsequently, the fragmented DNA was processed using the Ion DNA Barcoding kit (Life Technologies, Carlsbad, CA, USA) and its size selected using the E-Gel SizeSelect 2% Agarose kit (Life Technologies). The size distribution of the DNA fragments was analysed using the High Sensitivity Kit (Agilent, Santa Clara, Santa Clara, CA, USA). Further sample processing was performed using the Ion OneTouch Kit (Life Technologies). Finally, the amplified DNA was sequenced using the 318 chip (Life Technologies). Raw reads were assembled de novo using Assembler SPAdes software [7 (link)]. The genome was annotated using the RAST (Rapid Annotations using Subsystems Technology) database [8 (link),9 (link)].

Genomic DNA was extracted from 10-μm-thick sections of 10% neutral FFPE adenoma tissue samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany).
Targeted gene sequencing was performed as previously described [15 (link)]. Ten nanograms of DNA was used for multiplex PCR of covered coding regions in the KCNJ5, ATP1A1, ATP2B3, and CACNA1D genes (Ion AmpliSeq panel; Life Technologies, Grand Island, NY, USA). Fragment libraries were constructed by DNA fragmentation, barcode and adaptor ligation, and library amplification using an Ion DNA Barcoding kit (Life Technologies) according to the manufacturer’s instructions. The size distribution of the DNA fragments was analyzed on an Agilent Bioanalyzer using a High Sensitivity Kit (Agilent, Santa Clara, CA, USA). Template preparation, emulsion PCR, and ion sphere particle (ISP) enrichment were performed using an Ion Xpress Template kit (Life Technologies) according to the manufacturer’s instructions. The ISPs were loaded onto a P1 chip and sequenced using an Ion P1 sequencing kit (Life Technologies).