E220 focused ultrasonicator

200 ng DNA were quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and sheared on the Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) using the 8 microTUBE–50 Strip AFA Fiber V2 following manufacturer’s instructions. The treatment time was optimized for FFPE material. The treatment settings were the following: Peak Incident Power (W): 175; Duty Factor: 10%; Cycles per Burst: 200; Treatment Time (s): 200; Temperature (°C): 7; Water Level: 6. For DNA library preparation and enrichment the NEOplus v2 RUO kit (NEO New Oncology, Cologne, Germany) was used following manufacturer’s instructions with 100 ng DNA input. Post-enriched libraries were quantified, pooled and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA).
Quality of the NextSeq 500 (Illumina) sequencing runs were assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data were analyzed with the NEOonsite Data Analysis RUO (version 1.4.1) and the NEO software NEOdb 2.2 (NEO New Oncology).

Pre-treatment biopsy specimens were obtained by incisional biopsy (62 cases) or surgical resection (29 cases). TMB was measured by TruSight Oncology 500 (Illumina Inc., San Diego, CA, USA) as described previously [22 (link)]. Briefly, 40 ng of DNA was quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Inc., Waltham, MA, USA) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and then sheared using a Coraris E220 focused-ultrasonicator (Woburn, MA, USA) and an 8 microTUBE–50 Strip AFA Fiber V2 following the manufacturer’s instructions. For DNA library preparation and enrichment, the TruSight Oncology 500 Kit (Illumina) was used following the manufacturer’s instructions. Post-enriched libraries were quantified, pooled, and sequenced on a NextSeq 500 (Illumina). The quality of the NextSeq 500 (Illumina) sequencing runs was assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data were analyzed with the TruSight Oncology 500 Local App Version 1.3.0.39 (Illumina). TMB was reported as mutations per megabase (Mb) sequenced, and high TMB was defined as more than 10 mutations per Mb (≥10 Mut/Mb).

40 ng DNA were quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) and sheared on the Covaris E220 Focused-ultrasonicator (Woburn, MA, USA) using the 8 microTUBE–50 Strip AFA Fiber V2 following manufacturer’s instructions. The treatment time was optimized for FFPE material. The treatment settings were the following: Peak Incident Power (W): 75; Duty Factor: 15%; Cycles per Burst: 500; Treatment Time (s): 360; Temperature (°C): 7; Water Level: 6. For DNA library preparation and enrichment the TruSight Oncology 500 Kit (Illumina) was used following manufacturer’s instructions. Post-enriched libraries were quantified, pooled and sequenced on a NextSeq 500 (Illumina Inc., San Diego, CA, USA).
Quality of the NextSeq 500 (Illumina) sequencing runs were assessed with the Illumina Sequencing Analysis Viewer (Illumina). Sequencing data was analyzed with the TruSight Oncology 500 Local App Version 1.3.0.39 (Illumina).