PTPRT mutational screening in an independent series of familial/early-onset non-polyposis CRC cases
Mutational PTPRT screening in 473 familial/early-onset MMR-proficient non-polyposis CRC patients was performed using a combination of PCR amplification in pooled DNAs and targeted NGS (Puente et al., 2011). DNA pools were obtained adding equimolecular quantities of each sample (48-96 samples/pool), and used as templates for PCR amplification of each region of interest (i.e. coding exons +/- 20bp) using the Phusion High-Fidelity DNA Polymerase (New England Biolabs. Primers are available upon request. PCR products were purified (QIAquick PCR Purification Kit, Qiagen,), quantified (NanoDropTM, Thermo Fisher Scientific) and mixed in equimolecular quantities. These were ligated and used for paired-end library preparation for subsequent sequencing in a HiSeq 2000 (Illumina) at Centro Nacional de AnĂ¡lisis GenĂ³mico (CNAG, Barcelona, Spain). Details of the data analysis are shown in Terradas et al. 2019 (Terradas et al., 2019). Variant-specific KASP genotyping assays (LGC Genomics) and Sanger sequencing were used for validation and identification of the carriers of each variant at STAB VIDA (Caparica, Portugal) and Macrogen (Amsterdam, the Netherlands). Data was analyzed with SeqMan Pro (DNASTAR Lasergene 13).