Ion ampliseq designer software

The panel was designed by referring to the previous studies with Ion AmpliSeq designer software (Thermo Fisher Scientific).¹⁹, ²⁰ Seventy significantly mutated genes (SMGs) were included to determine the EC panel in‐house (Table S1). This panel covers SMGs of both ESCC and esophageal adenocarcinoma. The SMGs were selected according to the following criteria: (a) genes often involved in EC, obtained from TCGA and other projects.¹⁴, ¹⁵, ¹⁶, ¹⁷, ²¹ (b) genes frequently mutated in EC, referring to the COSMIC database (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic). Finally, the panel contained 4410 primer pairs.

Ion AmpliSeq™ Designer software (Thermo Fisher Scientific Inc.) was used to design primers that covered the exons of 53 lung cancer-related genes (Supplementary Table S4) reported by The Cancer Genome Atlas Project [19 (link), 20 (link)]. Targeted deep sequencing was performed as previously described [21 (link)]. Briefly, sequencing libraries were prepared using the Ion AmpliSeq™ Library Kit 2.0 (Thermo Fisher Scientific Inc.). Emulsion PCR was performed using the Ion OneTouch system and Ion PI™ Template OT2 200 Kit v3 (Thermo Fisher Scientific Inc.). Template-positive Ion Sphere™ Particles were enriched using the Ion OneTouch system and loaded onto an Ion PI Chip v2. Massively parallel sequencing was performed using an Ion Proton™ instrument and the Ion PI™ Sequencing 200 Kit v3 (Thermo Fisher Scientific Inc.). Single nucleotide variants, insertions, and deletions were annotated using the Ion Reporter Server System (Thermo Fisher Scientific Inc.). Lymphocytes from peripheral blood DNA were used as controls to detect variants in tumors [22 (link)]. Mutations with a variant allele fraction of ≥1.0% were considered confidence-based somatic mutations; these were also examined in bronchial wash fluid, sputum, and plasma specimens. Sequencing data were visualized using Integrative Genomics Viewer [23 (link)].

Amplification primers were designed to cover the exonic regions of five genes, GRIK1 (GeneID:2897), GRIK2 (GeneID:2898), GRIK3 (GeneID:2899), GRIK4 (GeneID:2900), and GRIK5 (GeneID:2901), with the Ion AmpliSeq™ Designer software (Thermo Fisher Scientific Inc., Waltham, MA, USA). Ion semiconductor sequencing, PCR reactions, and fluorescence-based cycle sequencing were performed following previously described methods [41 (link)].

A custom, single pool, multiplexed, PCR-based, NGS library panel was designed using Ion AmpliSeq designer software (Thermo Fisher Scientific, Waltham, MA, USA) to target 97 specific regions from 94 genes for a total panel size of 15.58 Kb per library^{34 (link)}. The information on the genomic coordinates of the amplicons included in the panel can be found in Supplementary Table 1. The Libraries were constructed using Ion AmpliSeq^TM Library Kit v2.0 (Life Technologies), according to the manufacturer’s instructions^{34 (link)}. To simply explain, 10 ng of DNA from either frozen, non-WGA FFPE or FFPE WGA products were amplified for 21 cycles, followed by FUPA treatment and ligation of Ion Xpress plus Universal adaptors. The libraries were purified with Agentcourt Ampure XP beads (Beckman Coulter, Fullerton, CA) and re-suspended in 1× low TE buffer. The libraries were quantified by using the Ion Library TaqMan™ Quantitation Kit (Thermo Fisher Scientific; Cat. No. 4468802).

Multiplex primer pools were designed using Ion AmpliSeq Designer software (Thermo Fisher Scientific, MA, USA). This “on demand panel” is composed by 45 genes causative of dysglicemia and its complications (Supplementary Table 1) and it covers 100% of the coding region.
Genomic DNA was extracted from EDTA whole blood using QIAamp DNA Blood Midi kit (Qiagen GmbH, Hilden, Germany) and purified with Amicon Ultra 0,5 mL (Merk Millipore LTD, IRL). Eluted DNA was quantified with Nanodrop Spectrometer Thermo Fisher Scientific, MA, USA. Enrichment of exonic sequences was performed with an Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific, MA, USA) and sequenced on an Ion PGM (Thermo Fisher Scientific, MA, USA) using 316 Chip (Thermo Fisher Scientific, MA, USA) following the standard manufacturer’s protocol.
Sequencing data were analyzed with Coverage Analysis and Variant Caller plugins available within the Ion Torrent Suite software TS 5.18 and contextually with IonReporter. All the pathogenic or likely pathogenic variants detected by Ion PGM were amplified using specific couple of primers and confirmed by direct sequencing of the PCR products.

For RNA sequencing, PCR primers were designed using the Ion AmpliSeq Designer software program (Life Technologies). The Ion AmpliSeq RNA Library Kit (Life Technologies) was used to construct the RNA library according to the manufacturer's instructions. Briefly, 20 ng of total RNA were reverse transcribed with the SuperScript III enzyme, followed by PCR amplification. The Ion Xpress Barcode adapters (Life Technologies) were ligated into the PCR products and purified with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Purified libraries were pooled and sequenced on an Ion Torrent PGM (Life Technologies) using the Ion PGM 200 Sequencing Kit v2 and the Ion 318 v2 Chip Kit.

Ion AmpliSeq Designer software (Life Technologies, Carlsbad, CA, USA) was used for the design and synthesis of a multiplexed gene panel encompassing 265 PID genes. The list of PID genes screened for is provided in Table S1 in Supplementary Material. Primers were optimized to provide amplicons (200 bp) with at least 90% coverage of coding sequence and a minimum of 10 bp flanking regions of associated introns. Ten nanograms of extracted DNA samples were sufficient to generate the Ion Proton AmpliSeq library. Details of library preparation, NGS, data processing, bioinformatics analysis, workflow of detecting point mutations, and copy number variants (CNVs) were previously described (28 (link)). The assay has an overall sensitivity of 96% for single nucleotide variants (SNVs) and 92% for detecting Indels. CNV analyses have a sensitivity of 100% for detecting large homozygous deletions (28 (link)).