TP53 Mutation Detection by Targeted Sequencing

The targeted massive parallel sequencing of the tumour DNA generated data on the TP53 mutation status. The fragmentation of 1,000 ng dsDNA was achieved using the Covaris® M220 Focused-ultrasonicator™ (Covaris, Woburn, MA, USA). The library preparation was performed using the Agilent SureSelectXT reagent kit (Agilent Technologies, Santa Clara, CA, USA), and the individual samples were run on a MiSeq instrument (Illumina, San Diego, CA, USA). This design included +/−10 nucleotides at exon-intron borders, to cover potential splice site mutations. The TP53 data was extracted from a sequencing effort applying baits targeting 360 genes as previously described in detail^{37 (link)}. Preliminary mutation calling was performed using the MiSeq Reporter (MSR) software, and the raw mutation calling output was revised by the application of post-processing filters. All of the suspected TP53 mutations were validated by the manual inspection of the sequencing reads using the Integrative Genomics Viewer^{38 (link)}.