Twist human core exome kit

Exome sequencing was performed according to local protocols, namely, standard chromosome analysis on peripheral blood lymphocytes. Microdeletions were identified by array-CGH (Agilent, Santa Clara CA, USA) or SNP-array (Beadchip 850K, Illumina, San Diego, CA, USA) platforms at a resolution of 100 kb. Comprehensive open reading frame/splice site mutational analysis of clinical exomes were conducted through the Twist Human Core Exome Kit and sequenced on the Illumina NovaSeq6000 Platform. Lastly, an amplification study of the CGG repeats of the FMR1 gene for X fragile syndrome was carried out using RP-PCR. Further information is available upon request.

TP53 mutational status was assessed using whole-exome sequencing (WES, n = 90) or Sanger sequencing of exons 5 to 8 (n = 5). WES data were generated using Illumina Nextera Exome enrichment (n = 89) or TWIST Human Core Exome kit (n = 1) and sequenced on an Illumina NovaSeq within the Newcastle University Genomics Core Facility or Illumina HiSeq by Eurofins Genomics (Germany). Data were analysed using the Genome Analysis Toolkit (GATK 3.7) and variants called using Mutect2. PCR products for Sanger sequencing were amplified using primers designed for TP53 (Supplementary Table 1) and sequenced by Eurofins Genomics. WES base calls were confirmed by Sanger sequencing in 39 cases, with 100% concordance between sequencing methods.
Copy number alterations (CNAs) of 17p and other chromosomes were identified using Affymetrix Cytoscan HD, Genome-wide Human SNP Array 6.0 or OncoScan arrays performed by Eurofins Genomics. Raw data were analysed and visualised in Nexus Copy Number 10.0 (BioDiscovery) to detect CNAs and copy number neutral loss of heterozygosity (CNN-LOH) in all samples.