Le220 focus ultra sonicator

Whole exome sequencing libraries were prepared using the SureSelect XT library preparation kit (Agilent). DNA was sheared using a LE220 Focus Ultra-sonicator (Covaris), and the fragments were end-repaired, adenylated, ligated to Illumina sequencing adapters, and amplified by PCR. Exome capture was performed using the SureSelect XT v4 51Mb capture probe set (Agilent) and captured exome libraries were enriched by PCR. Final libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems), Qubit Fluorometer (Life Technologies) and 2100 BioAnalyzer (Agilent), and were sequenced on a HiSeq2500 sequencer (Illumina) using 2 x 125bp cycles.

DNA was extracted using the Blood and Tissue Kit (Qiagen). DNA quality and integrity was then analyzed using BioAnalyzer (Agilent Technologies). WES libraries were prepared with the SureSelectXT Library Preparation Kit (Agilent) according to the manufacturer’s instructions. Extracted DNA was then sheared using an LE220 Focus-ultrasonicator (Covaris), and the fragments were end-repaired, adenylated, ligated to Illumina sequencing adapters, and amplified by PCR. Exome capture was performed using the SureSelectXT v4 51 Mb capture probe set (Agilent), and captured exome libraries were then enriched by PCR. Final libraries were quantified with the KAPA Library Quantification Kit (KAPA Biosystems), Qubit Fluorometer (Life Technologies), and 2100 BioAnalyzer (Agilent) and were sequenced on a HiSeq2500 sequencer (Illumina) using 2 × 125 bp cycles as previously described^{8 (link)}. Tumor mutational burden was calculated as the number of coding mutations per megabase, also incorporating additional exome data from previously published WES samples from our laboratory analyzed using the same mutation calling algorithm^{5 (link),8 (link),10 (link)}.