Ion pgm

Next-generation sequencing was performed to detect single nucleotide variants (SNVs), short insertions, and deletions (indels). We investigated mutations of the SDHA, SDHB, SDHC, SDHD, fumarate hydratase (FH) and rearranged during transfection (RET) genes by sequencing the coding exons and intron flanking regions in tumor specimens, as described previously [26 (link)]. The custom primers for these regions were designed with Ampliseq Designer (Life Technologies). Library construction and sequencing were performed with an Ion AmpliSeq Library Kit 2.0, Ion PGM IC 200 kit, and Ion PGM (Life Technologies), according to the manufacturer's instructions. Sequencing data were analyzed with Torrent Suite, and variant call was conducted with Torrent Variant Caller, Ion Reporter (v.5.1.0). Ion AmpliSeq panels cover broad research areas for germline analysis, including genes recommended by the American College of Medical Genetics and Genomics [27 (link)]. Then, the accuracy of the Ion Torrent sequencer platform in detecting SDHB gene mutations was confirmed according to a previously published method [28 (link)].

FFPE tumor samples (n = 25 for mCRC and n = 20 for NSCLC) were microdissected (microdissector LMD2000, Leica, Germany, EU) and DNA was purified from the areas of the samples with the highest percentage of tumor cells using the QIAamp DNA FFPE Tissue kit (Qiagen, Cat No./ID: 56404, Valencia, CA, USA) as per manufacturer's instructions. These samples were then analyzed using a customized Ampliseq library and next-generation sequencing (NGS) on the Ion PGM (Life Technologies, Carlsbad, CA, USA).
Plasma was prepared from 30 mL of blood collected in K₂ EDTA tubes (BD, 367525, 18 mg). All blood samples were delivered to the laboratory within 24 hours after collection. Detailed pre-analytical considerations have been previously published [13 (link)]. The haploid GE corresponds to the haploid Genome Equivalent (330 GE blood DNA for 1 ng/μL cfDNA).

DNA was extracted from frozen fecal samples using the DNA stool Mini kit (Qiagen), following the manufacturer’s instructions. DNA concentrations were measured using QuBit 2.0 Fluorometer kit (Life Technologies). Bacterial 16S ribosomal RNA (rRNA) genes contain nine “hypervariable regions” (named from V1 to V9) that demonstrate considerable sequence diversity among different bacteria. These regions of bacterial 16S rRNA genes were amplified by PCR using two sets of primers V2-4-8 and V3-6,7–9 available in the Ion 16S Metagenomics kit (Life Technologies). The PCR products were purified using SPRI method (Solid Phase Reversible Immobilization) (Agencourt AMPURE XP). Each amplicon was then assessed for fragment size distribution and DNA concentration using a Bioanalyser 2100 (Agilent Technologies, USA). Library preparation followed the Ion Plus Fragment Library (Life Technologies). Products were first end-repaired and purified using SPRI method (Agencourt AMPURE XP). Then, library was bar-coded using the Ion Xpress Barcode Adapter 1–16 kit (Life Technologies) and sample was again purified using SPRI method. Finally, emulsion PCR and enrichment steps were carried out using the Ion PGM (Life Technologies). Sequencing was undertaken using 316 chips and Ion PGMSequencing 400 on the Ion Torrent Personal Genome Machine (PGM).

Tumor tissue samples of the 50 patients were used to investigate mutations of the Nrf2, Keap1, von Hippel-Lindau (VHL), and fumarate hydratase (FH) genes. We performed targeted next-generation sequencing of the coding exons and intron flanking regions of these four genes [19 (link)], using customized primers designed with Ampliseq Designer (Life Technologies). Construction of a library and sequencing were performed with an Ion AmpliSeq Library Kit 2.0, Ion PGM IC 200 kit, and Ion PGM (Life Technologies) according to the directions of the manufacturer. Raw data from each sequencing reaction were analyzed with Torrent Suite version 4.2.1.

MeDIP libraries were templated using the Ion OneTouch 2 System instrument with either the Ion PGM Template OT2 200 kit for the PGM instrument or the Ion PI Template OT2 Kit v2 for the Proton instrument according to manufacturer’s protocols (Life Technologies). Following template reactions, templated libraries were assessed for quality using the Ion Sphere Quality Control Kit (Catalog No. 4468656) and Qubit fluorometer (Life Technologies). Templated libraries that passed manufacturer recommended criteria were enriched using the Ion OneTouch ES instrument. Immediately after enrichment, MeDIP libraries were sequenced on either an Ion PGM or Ion Proton Semiconductor Sequencing machine (Life Technologies).