Biorupter ultrasonicator

A focus of tumor with higher than 70% tumor cellularity was marked on an H&E-stained section. Matching areas of the tumor were macrodissected for DNA sequencing. Normal squamous epithelium from the excised skin was used as a matched control. Briefly, we used a KAPA HyperPrep library preparation kit (Kapa Biosystems, Wilmington, MA, USA) to prepare indexed libraries from 200 ng genomic DNA that had been sheared using a Biorupter Ultrasonicator (Diagenode, Denville, NJ, USA). The multiplexed library pool was hybridized to the MD Anderson T200.1 probe pool (Roche NimbleGen, Madison, WI, USA). Following hybridization and reaction cleanup, the enriched libraries were amplified with seven cycles of postcapture PCR, then assessed for target enrichment by quantitative PCR. Sequencing was performed in one lane of a HiSeq4000 Sequencer (Illumina, San Diego, CA, USA) using a 150 bp paired-end configuration.

Briefly, indexed libraries were prepared from 500 ng of Biorupter Ultrasonicator (Diagenode, Denville, NJ, USA)-sheared, genomic DNA using the KAPA Hyper Library Preparation Kit (KAPABiosystems, Wilmington, MA, USA). The indexed libraries were prepared for capture with six cycles of pre-ligation-mediated PCR amplification. Following amplification and reaction cleanup, the libraries were quantified using the Qubit^™ dsDNA HS Assay (ThermoFisher, Waltham, MA, USA) and assessed for size distribution using the Fragment Analyzer (Advanced Analytical, Ames, IA, USA). Library concentrations were normalized, and the libraries were multiplexed 8 libraries/pool. Each multiplexed library pool was hybridized to a probe pool from the SeqCap EZ Human Exome Enrichment Kit v3.0 (Roche-NimbleGen, Madison, WI, USA). The enriched libraries were amplified with eight cycles of post-capture PCR, then assessed for exon target enrichment by qPCR. The exon-enriched libraries were then assessed for size distribution using the Fragment Analyzer (Advanced Analytical) and quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems). Sequencing was performed on the HiSeq4000 Sequencer (Illumina, San Diego, CA, USA), one capture (eight samples) per lane using the 150 bp paired-end configuration.