Ion torrent s5

Several well-characterized human NB cell lines were generously provided by Drs. David Barrett (University of Pennsylvania, Philadelphia, PA, USA), Patrick Reynolds (Texas Tech), and Peter Houghton (University of Texas, San Antonio, TX, USA) [42 (link),43 (link),44 (link),45 (link),46 (link)]. Cells were cultured in standard medium (Eagles Modified, DMEM, or RPMI; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, penicillin/streptomycin, 1% L-glutamine, 2% Gibco HEPES buffer, 1% non-essential amino acids, and 0.02% 2-mercaptoethanol (all media components from Thermo Fisher Scientific). The previously reported genomic lesions in each cell line were confirmed by targeted sequencing (Table 2). Briefly, library preparation and targeted sequencing using the Oncomine Childhood Cancer Research Assay (OCCRA, Thermo Fisher Scientific) were performed on Ion Chef and Ion Torrent S5 platforms (Thermo Fisher Scientific) following the manufacturer’s protocols. The average read depth for the OCCRA panel was 5–7 × 10⁶ per sample for DNA and 1–2 × 10⁶ for RNA. SNVs were retrieved with Ion Reporter software (version 5.2). Copy number measurements were retrieved with Ion Reporter software (version 5.2) for genes with >5 probes, including those that were validated for copy number gains as described elsewhere [47 (link)].

Peripheral blood samples were harvested at diagnosis from BC or OC patients through a vacutainer syringe containing EDTA. Genomic DNA was isolated using the DNeasy^® Blood Kit (QIAGEN, Hilden, Germany). After the extraction phase, DNA has been quantified by Qubit^®3.0 fluorometer (Thermofisher Scientific, Waltham, MA, USA) and its quality has been assessed through the use of 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
The genetic analysis for BRCA1/2 was performed as previously described (4 (link), 26 (link)).
Sequencing analysis was performed using Ion 520 Chip (Thermofisher Scientific, Waltham, MA, USA) and Ion Torrent S5 (Thermofisher Scientific, Waltham, MA, USA) NGS platform. Obtained data were analyzed using both Amplicon Suite (SmartSeq s.r.l.) and Ion Reporter Software v.5.12 (Thermofisher Scientific, Waltham, MA, USA). NGS data analysis was performed with the standardization of sequencing coverage depth in order to minimize the probability of false positive and negative results in clinical practice, considering a minimum coverage of 500× to each sample.

The mutational profile of 32 recurrently mutated genes in myeloid malignancies was determined using an Illumina TruSeq Custom Amplicon next-generation sequencing gene panel (Supplementary Table 2) and the TruSeq Amplicon 2.0 BaseSpace app workflow [51 (link)] (Illumina, San Diego, CA, USA). 31 inositide-specific point mutations and small indels (Supplementary Table 3) were examined using the Ion Torrent S5 with an Ion AmpliSeq™ On-demand Panel (Thermo Fisher Scientific, Whaltam, MA, USA). Sequencing alignment was viewed by the Integrative Genomics Viewer Software (Broad Institute, Cambridge, MA, USA) using the Human Genome Build 19 (Hg19) as reference [52 (link)]. Further details can be found in the Supplementary Information.

In the validation phase, a minimum of 10 ng of total RNA was converted to cDNA using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). In the clinical phase, when the analysis was successfully performed by the conventional method, we conducted an NGS assay. An Ion Torrent adapter‐ligated library was generated using the custom panel described above. The concentration and size of the library were determined using the Applied Biosystems StepOne Real‐Time PCR system and Ion Library TaqMan Quantitation kit (both from Thermo Fisher Scientific). Sample emulsion PCR, emulsion breaking and enrichment were performed using the Ion Chef system (Thermo Fisher Scientific), according to the manufacturer's instructions. An input concentration of one DNA template copy per ion sphere particle (ISP) was added to the emulsion PCR master mix, and the emulsion was generated using the Ion Chef system (Thermo Fisher Scientific). Template‐positive ISPs were enriched, and sequencing was performed using semiconductor chips on the Ion Torrent S5 (Thermo Fisher Scientific). In this study, we used semiconductor chips of Ion 530 Chip Kit, which was later we changed to the Ion 540 Chip Kit, in order to collect an adequate number of sequenced reads.