Ion reporter software v5

Variants were catalogued through Ion Reporter software v.5.2 (ThermoFisher, USA). Functional prediction was performed using MutationTaster^{46 (link)}, SIFT^{47 (link)}, PolyPhen-2^{48 (link)}, SILVA^{49 (link)}, RNAFold^{50 (link)}, miRbase^{51 (link)}, Human Splicing Finder 3^{52 (link)}, PredictSNP-2^{53 (link)}, ApasDB^{54 (link)}, EnhancerAtlas^{55 (link)}, MethPrimer^{56 (link)}, and Haploview v.4.2^{57 (link)}, according to the developers instructions. Packages motifbreakR^{58 (link)} and biomaRt^{59 (link)} were integrated in RStudio v.3.3 for the functional predictions.

Analysis of the sequencing data was performed using Torrent Suite Software v5.2.2 (Thermo Fisher Scientific). This workflow was created by adding a custom hotspots Browser Extensible Data file to report mutations of interest, and a custom CNV baseline using the manufacturer’s default workflow as described previously [34 (link),35 (link)]. The pipeline included signaling processing, base calling, quality score assignment, adapter trimming, read mapping to the human genome assembly GRCh37, quality control of mapping, coverage analysis with downsampling, and variant calling. The identification of variants was performed using the Torrent Variant Caller plug-in and Ion Reporter Software v5.2 (Thermo Fisher Scientific), and coverage maps were generated using the Coverage Analysis plug-in (Thermo Fisher Scientific). Additionally, ANNOVAR was used for functional annotation of identified single nucleotide polymorphisms (SNPs) to investigate their genomic locations and variation [36 (link)]. To eliminate error artifacts, sequence data were visually confirmed using the Integrative Genomics Viewer (Broad Institute, Cambridge, MA, USA). This workflow was able to report SNPs and indels in as low as 1% of the variant allele fraction. Based on the results of a feasibility study, the variant allele fraction threshold was established at 5%.

Next-generation sequencing was performed for WES analysis using the Ion S5™ Sequencer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). The Ion AmpliSeq™ Exome RDY kit (Thermo Fisher Scientific, Inc., Wilmington, DE, USA) was used, according to the manufacturer’s protocol. The Ion reporter software v.5.2 (Thermo Fisher Scientific) was used to analyse the mutations (https://ionreporter.thermofisher.com/ir/). Under an assumed autosomal recessive mode of inheritance, all variants were assessed individually according to the clinical phenotype, minor allele frequency (MAF) score and pathogenicity scores were calculated using prediction programmes. Variants were filtered to retain non-synonymous changes with a MAF of <0.01 using combined datasets from the 1000 Genomes Project, the Exome Variant Server project and the Genome Aggregation Database (GnomAD). The potential functional impact of the disease candidate variants were assessed using SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.mutationtaster.org/) and VarSome (https://varsome.com/). All genetic variants were screened for pathogenicity, mode of inheritance and clinical phenotypes. Finally, candidate pathogenic variants identified by WES were verified with Sanger sequencing.

The Oncomine Focus Assay (Thermo Fisher Scientific) was used for the detection of gene fusions in 23 genes including ALK, ROS1, RET, BRAF, MET, FGFR1-3, NTRK1-3. 10–50 ng of DNase digested RNA was used for library preparation according to manufacturer’s instructions. Final libraries were diluted, pooled and further processed on Ion spheres using Ion 520 or 318 Chef Kits (Thermo Fisher Scientific) on the Ion Chef (Thermo Fisher Scientific) or the Ion 520 & Ion 530 Kit-OT2 (Thermo Fisher Scientific). Sequencing was performed on Ion S5 System or PGM systems (Thermo Fisher Scientific) using the Ion 520 or 318 Chips (Thermo Fisher Scientific). Sequencing data were analyzed with the Ion Reporter Software v5.10 (Thermo Fisher Scientific). A fusion was classified as present with greater than 20 reads providing evidence for the fusion and/or an 3′/5′ imbalance value thresholds for strong evidence of a fusion: ALK: ≥ 0.0015, RET: ≥ 0.55, ROS1: ≥ 2.1. The 3′/5′ imbalance score calculates the differences in expression between the 5′ assay and the 3′ assay of each gene. In the case of a fusion event, the 3′ portion of the gene is overexpressed in comparison to the 5′ assay. Resulting in a 3′/5′ imbalance score greater than the baseline.
The time for analysis heavily relied on the used hardware. On the specific server hardware delivered with the system one sample took around 15 min.