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).