Applied biosystems sanger sequencing kit

For TP53 sequencing of PT-res pool cells, genomic DNA was extracted from cultured cells using Maxwell 16 DNA purification kit (Promega). Then, 50 ng of genomic DNA was amplified with TruSeq Custom Amplicon kit (TSCA, Illumina, San Diego, CA, USA) specially designed for the targeted sequencing of TP53 (13 amplicons) among others. Libraries were run in an Illumina MiSeq instrument achieving a median coverage >1000 reads. Data were aligned to human reference genome hg18 and analyzed, after quality control, using Variant Studio and the IGV program, reporting only variants with a mutant allelic frequency (MAF) greater than 5%. We considered not only variants inside the coding sequence but also the ones inside the 5′-, 3′-untranslated regions (UTRs) and inside splicing regulatory elements [17 (link)] (http://p53.iarc.fr/TP53GeneVariations.aspx). Validation of NGS data was performed using the DNA Sanger sequencing method using the Applied Biosystems™ Sanger Sequencing Kit and the ABI3130xl instrument (Applied Biosystems, Waltham, MA, USA).

The presence of the p.Ser1600Gly variant was corroborated by Sanger sequencing. Given the position of the variant, the PCR primers were designed to amplify a portion of the second bromodomain of the TAF1 gene. Sanger sequencing was performed using an Applied Biosystems Sanger Sequencing Kit (Thermo Fisher Scientific). DNA sequencing was run on an Applied Biosystems 3730XL DNA Analyzer (Foster City, CA). The user can expect up to 600 bases of reliable sequence per read in one direction. The sequencing data were loaded into the ABI Sequence Scanner Software v1.0 for further analysis and genotype calling. All sequence traces were also manually reviewed to ensure the reliability of the genotype calls.