Ion torrent technology

Glioma tissues samples (Table S1) were obtained from surgeries at Hospital 12 de Octubre (Madrid, Spain), after patient’s written consent and with the approval of the Ethical Committee (Comité de Etica de la Investigación (CEI) del Hospital 12 de Octubre) (CEI 14/023). Mutations and copy number variations in TP53, PTEN, and EGFR were identified by using a next-generation sequencing (NGS) panel (Ion Torrent technology, ThermoFisher Scientific, Waltham, MA, USA) [33 (link)].

VviAGL11 gene sequences (promoter and CDS) were amplified from genomic DNA of table grapevine varieties by using the Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific). PCR products were purified from agarose gel using Illustra GFX PCR DNA and Gel Band Purification Kit (Merck KGaA, Darmstadt, Germany) and then sequenced by Ion Torrent technology (Thermo Fisher Scientific). Amplicon libraries were prepared by using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific) following manufacturer instructions. The enriched library was loaded on Ion PGM 314 chip and sequenced on the Ion PGM™ sequencer using Ion PGM™ Sequencing 200 Kit v2. Reads were aligned on the grapevine reference genome (12X.v2) of Pinot Noir [43 (link)]. The depth of sequencing obtained was approximately 800X. Variant caller plugin provided in the Torrent Suite Software was run for the identification of polymorphisms across the reference. Sequences were viewed by the Integrative Genomics Viewer (IGV) [44 (link)].

Libraries were prepared using Ion Torrent technology and Ion Chef workflows (Thermo Scientific, Waltham, MA, USA). Sequencing was performed in the S5XLS system, and analysis of the raw sequencing data was conducted by Ion Torrent Suite v.5.10.0, according to the manufacturer’s instructions. Sequencing of the D1644 isolate performed twice to resolve ambiguities related to the presence of an IncN plasmid type harboring bla_NDM-1. The detection of the plasmid type and downstream analysis for D1644 performed on the merged sequencing products. Raw sequencing data were quality-checked and trimmed to keep only the reads that match the minimum length and quality criteria. Taxonomic assignment was performed as a quality step to filter out samples that may have been contaminated by foreign DNA during sample preparation and to verify the presence of the isolated species. Kraken2 was used for the taxonomy classification built on the standard database that includes NCBI taxonomic information, complete RefSeq microbe genomes, human genomes, and a vector collection [25 (link)]. Genomes were de novo assembled by AssemblerSPAdes and SPAdes using the default settings for the k-mers and the Ion Torrent parameter [26 (link)].

The NGS gene panel was designed to be specific for EB, including the vast majority of cases of the disease at a viable cost for the Brazilian reality. Thus, the 11 main genes associated with the four types of EB were included in the panel (KRT5, KRT14, PLEC, TGM5, LAMA3, LAMB3, LAMC2, COL17A1, ITGB4, COL7A1, and FERMT1). The 62.72-kb and 574-amplicon panel was designed using the AmpliSeq Designer tool (Thermo Fisher Scientific – United States). Ion Torrent technology (Thermo Fisher Scientific – United States) was used for sequencing. Information about the running parameters, data analysis, investigation, classification, and details of the identified variants were described in a previous study.¹³ (link)

Whole exome sequencing was performed for nine patients' DNA samples using Ion Torrent technology (Thermo Fisher Scientific). Libraries were prepared following the protocol described in Ion AmpliSeq Exome RDY library preparation user guide (Publication number MAN0010084) using Ion AmpliSeq Library Kit Plus (Cat. No. 4488990). One hundred nanograms of genomic DNA was used in library preparation with Ion AmpliSeq Exome RDY Kit 1×8 (Cat. No. A38262); then, the libraries were purified using a magnetic plate and subsequently quantified using Ion Library TaqMan quantitation kit (Part number 4468802) and Real-time PCR instrument 7500 Fast (Life Technologies, USA). Each sample was assigned a distinct barcode using an IonXpress barcode adapters 1–16 kit (Cat. No. 4471250). Barcoded libraries were diluted to 100 pM; three libraries were pooled and subjected to template preparation on Ion Sphere Particles (ISP) using the Ion OneTouch 2 System and the Ion PI Template OT2 Hi-Q Kit. Each templated ISP was loaded on the Ion PI v3 chip and sequenced on the Ion Proton instrument (Life Technologies, USA) using Ion PI Hi-Q sequencing Kit. Base calling and alignment were performed on a Torrent Suite v4.2 Server.

The nucleic acid extraction was conducted using either the Maxwell RSC Instrument (Promega, Madison, WI, USA) in combination with the Maxwell RSC FFPE Plus DNA kit or the Maxwell RSC RNA FFPE kit (Promega). After the extraction of nucleic acid, the concentration was measured by employing the Qubit Fluorometric quantification assay (Thermo Fisher Scientific, Waltham, MA, USA), utilizing the Qubit RNA HS Assay Kit and the Qubit dsDNA HS Assay Kit. The Ion Torrent™ Genexus™ Integrated Sequencer (Thermo Fisher Scientific) was used for the detection of genomic alterations by Ion semiconductor sequencing (Ion Torrent™ Technology, Thermo Fisher Scientific). The Oncomine™ Precision Assay GX panel (OPA) provided by Thermo Fisher Scientific was utilized, targeting 50 key genes. Among them, 45 genes were designed for DNA mutation detection, 18 for fusion detection, and 14 for copy number variant (CNV) detection. In addition, the panel incorporated a 5′/3′ expression imbalance strategy for the detection of novel fusions. By using this panel, up to 16 samples could be sequenced simultaneously on a single run with the Genexus sequencer.

Quantitative RNA expression analysis was performed by amplicon-based next-generation sequencing (NGS) using Ion Torrent technology (Thermo Fisher Scientific, Waltham, MA, USA). The Ion AmpliSeq™ RNA Library Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to construct the libraries from 20 ng of RNA. RNA was reverse transcribed, and targeted regions of RNA were PCR amplified using the Ion AmpliSeq™ RNA Cancer Panel (Thermo Fisher Scientific, Waltham, MA, USA) consisting of specific primers sets to amplify 50 target genes. (Additional file 1: Table S1) The Amplicons were then partially digested, and barcode adapters were ligated with the Ion Xpress™ Barcode Adapter Kit (Thermo Fisher Scientific, Waltham, MA, USA) to yield a barcoded library. Library concentrations were measured using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).
Templates for sequencing were prepared using the Ion PGM™ Hi-Q™ OT2 Kit (Thermo Fisher Scientific) and Ion OneTouch™ 2 System (Thermo Fisher Scientific, Waltham, MA, USA). Ion Sphere™ particles were enriched with Ion OneTouch ES (Thermo Fisher Scientific, Waltham, MA, USA) and loaded onto an Ion 318™ Chip (Thermo Fisher Scientific, Waltham, MA, USA).
Sequencing was performed on the Ion Torrent PGM™ System using the Ion PGM™ Hi-Q™ Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA).