Ampure purification system

Following collection of cells DNA was extracted using the DNeasy blood and tissue kit (Qiagen) using the standard procedure described in the handbook. For PCR amplification of the library 2 μg of DNA for each sample was used, with the DNA split into four 500 ng reactions. A one-step PCR approach was used with P5 and P7 primers. The P5 and P7 primer were comprised of the P5/P7 flow-cell attachment sequence, the Illumina sequencing primer and the vector-binding sequence (primer sequence in shown in Supplementary Table 2). In addition, to avoid the issue of reduced sequence diversity faced by amplicon libraries, PCR was performed with a mix of P5 primers containing a stagger region of different length. The PCR reaction was set-up on ice as follows: 10 μl ExTaq 10x reaction buffer (Takara), 8 μl dNTPs (10 mM), 0.5 μl P5 primer (100 μM), 1.5 μl ExTaq polymerase (Takara), 10 μl P7 primer (5 μM) and the reaction mix was made up to 100 μl with water. The PCR cycling parameters were as follows: 95°C 1 minute, 28 cycles (95°C 30 seconds, 53°C 30 seconds, 72°C 30 seconds), 72°C 10 minutes. The PCR product was then purified using the AMPure purification system (Beckmancoulter) ready for sequencing.

A TP53 variant, previously reported as pathogenic in ClinVar in relation to Li-Fraumeni syndrome and/or hereditary cancer pre-disposing syndrome, was found in P65 and validated using Sanger sequencing. Primers were designed using primer3 v0.4.0 (bioinfo.ut.ee/primer3-0.4.0/). SNPs in the primer-binding site were ruled out using the NGRL SNPCheck database (https://ngrl.manchester.ac.uk/SNPCheckV3/snpcheck) prior to ordering. PCR amplification was performed using the FASTstart High Fidelity PCR system (Roche, Madison, WI) at 59°C annealing temperature. Amplified PCR products were then purified using the AMPure Purification System (Beckman Coulter, Indianapolis, IN). The purified products were sequenced on the Applied Biosystems 3730 sequencer (Genomics Core at Einstein, NY). The Sequencer v4.0.1 software (Gene Codes, Ann Arbor, MI) was used to analyze sequencing files.