Ion pgm system

Full-length amplified cDNA amplicons and cDNA clones were sequenced by NGS at the DTU Multi-Assay Core (DMAC, Kgs. Lyngby, Denmark) using an Ion PGM system (Life Technologies, Carlsbad, USA). Consensus sequences were obtained by mapping the reads to the vKos reference sequence (KF977607.1) using CLC Genomics Workbench v.9.5.2 (CLC bio, Aarhus, Denmark). Consensus sequences were aligned using MAFFT in Geneious (Biomatters, Auckland, New Zealand). Low frequency single nucleotide polymorphisms (SNPs, > 0.5%) were identified for cDNA amplicons using a combination of BWA, Samtools, Lo-Freq-snp-caller, and SnpEff, as described previously [9 (link), 12 (link)]. Phylogeny was constructed using MrBayes v3.2.1 [13 (link), 14 (link)] on a full-length cDNA sequence alignment using the General Time Reversible (GTR) model with default parameters (nst = 6). The Markov chain Monte Carlo algorithm was run for 10,000,000 iterations, with a sampling frequency of 7000 using two independent runs with three chains each, in order to check for convergence. Burn in was set at 25% of samples. The consensus tree was visualized in FigTree v.1.4.3.

After obtaining informed consent from the patients or their parents, DNA was isolated from a whole-blood or heart tissue sample from each patient. Next-generation sequencing (NGS) was performed with 182 cardiac disorder-related genes (Table S1) using the Ion PGM System (Life Technologies, Carlsbad, CA, USA).
After all candidate pathogenic variants passed the selection criteria, to validate the result of NGS, Sanger sequencing was conducted.

The coding sequences of the EED, EZH2, and SUZ12 genes were analyzed using a targeted NGS approach. Experiments were performed on the NGS platform of the Cochin Hospital, Paris (Assistance Publique, Hopitaux de Paris, France). Briefly, the custom primer panel targeting the three genes (coding exons and IVS boundaries) was designed using AmpliSeq Designer (Life Technologies). For NGS library preparation, the Ion AmpliSeq 2.0 library kit was used according to the manufacturer's instructions. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Prior to library pooling and sequencing sample preparation, amplified libraries were quantified using the Qubit fluorometer system (Agilent Technologies). Emulsion PCR and enrichment were performed on the Ion OneTouch and Ion OneTouch ES instruments with the Ion PGM template OT2 400 and Ion PGM sequencing 400 kits (Life Technologies). The template-positive ion sphere particles were loaded on Ion 318 chips and sequenced with an Ion PGM system (Life Technologies). Sequence alignment and extraction of SNPs and short insertions/deletions were performed using the Variant Caller plug-in on Ion Torrent suite version 4.4 and Ion Reporter version 4.4 (Life Technologies). DNA sequences were visualized using the Integrated Genomics Viewer (version 2.3.3) from the Broad Institute.

DNA was amplified using Ion AmpliSeq™ Library Kit 2.0 (Life Technologies), then digested, and Ion Xpress™ Barcode adapters ligated and purified with Agencourt AMPure XP magnetic beads (Beckman Coulter). Libraries were quantified by qPCR using an Ion Library Quantification Kit (Life Technologies), templated on the Ion OneTouch2 System (Life Technologies) and sequenced on the Ion PGM System (Life Technologies). Reads were aligned by the PGM server with standard settings to the reference genome hg19; samtools v1.2 was used to calculate the on-target coverage.
IonReporter™ (v4.4) was used for mutation calling (parameters: Data Quality Stringency = 12, Downsample To Coverage = 4000, SNP/InDel/MNP Min Cov Each Strand = 50, SNP/InDel/MNP Min Variant Score = 15, SNP/InDel/MNP Min Coverage = 250, Hotspot Min Variant Score = 6, Hotspot Min Coverage = 150). All mutations called were manually reviewed in IGV and included in the analysis if they had a VAF ≥ 1%.

Viral RNA was extracted from clinical samples using the High Pure Viral RNA Kit (Roche Diagnostics, Tokyo, Japan). FMDV-specific genes were detected using the TaqMan Fast Virus 1-Step Master Mix (Life Technologies) with 900 nM of primer sets and 250 nM of probe targeting the 3D region [20 (link)]. In the present study, samples with a Ct value below 37 were defined as positive. The full length of the L-fragment gene of approximately 7.7 kb was amplified by RT-PCR using SuperScript IV One-Step RT-PCR System (Life Technologies) and two primer sets: set 1 consisted of a 5’-NT F primer (5’-CCGTCGTTCCCGACGTTAAAGGG-3’) and 2B331R primer (5’-GGCACGTGAAAGAGACTGGAGAG-3’) and set 2 consisted of a 2B217F primer (5’-ATGGCCGCTGTAGCAGCACGGTC-3’) and 3’-NT R primer (5’-CAATTGGCAGAAAGACTCTGAGGCG-3’). Their nucleotide sequences were analyzed using the Ion PGM system (Life Technologies).