Ion ampliseq tp53 panel

Analysis of the TP53 gene and the IGHV gene mutational status was performed on DNA extracted from purified pathological B cells using an automated extraction system (QIAsymphony SP, Qiagen, Hilden, Germany) based on the manufacturer’s reagents and protocols. The search for mutations in the TP53 gene was performed by Next-Generation Sequencing (NGS) with the Ion AmpliSeq TP53 Panel (Thermo Fisher Scientific, Waltham, MA, USA) on the Ion S5 sequencer (Thermo Fisher Scientific). The analysis of the results was performed using the Ion Reporter (Thermo Fisher Scientific) and Integrative Genomics Viewer (IGV) (v2.16.2, UC San Diego, CA, USA; and Broad Institute of MIT Harvard, Boston, MA, USA) software. Determination of the IGHV mutational status was performed by PCR/fragment analysis and Sanger sequencing, according to the ERIC (European Research Initiative on CLL) recommendations. Sequences were analyzed in IMGT/V-QUEST (v1.9.3, LIMG, Montpellier, France), to identify clonal rearrangements and to calculate their percent identity to the germline gene sequence. According to the ERIC recommendations, sequences with a percentage of identity lower than 98% were considered to be mutated while those with a ≥ 98% identity were reported as unmutated (27 (link)).

Genomic mutation of TP53 was studied by Next Generation Sequencing (NGS) using the Ion Ampliseq TP53 panel (ThermoFisher Scientific, Life Technologies, Les Ullis France). The design covers 100% of the coding sequence of TP53 including mutations at canonical splice sites and generates 24 amplicons on a length of 1 280 bases. Libraries were amplified by emulsion PCR and enriched using automatic system IonChef (ThermoFisher Scientific). Ion sphere particles were then sequenced with the Ion torrent personal genome machine (ThermoFisher Scientific) on 316v2 or 318v2 Chips (ThermoFisher Scientific) with high mean coverage > 3000X. Torrent Suite^™ version 5.0 software (ThermoFisher Scientific) was used to perform data analysis. Reads were mapped to the human hg19 reference genome. Data processing, alignment and mutation calling were performed using the Torrent Suite^™. The Variant Caller detected point mutations with a variant frequency ≥2% for Single Nucleotide Variation (SNV) and ≥5% for short insertion/deletion (INDEL). VCF files generated by Variant Caller were annotated by ANNOVAR [30 (link)].
In the 10 patients with TP53 mutation, BAM of wild-type samples for TP53 were also checked using Alamut Software (Interactive Biosoftware, Rouen, France).