Hamilton starlet liquid handling platform

After library construction, hybridization and capture are performed using the relevant components of IDT’s XGen hybridization and wash kit and following the manufacturer’s suggested protocol, with several exceptions. A set of 12-plex pre-hybridization pools are created. These pre-hybridization pools are created by equivolume pooling of the normalized libraries, Human Cot-1 and IDT XGen blocking oligos. The pre-hybridization pools undergo lyophilization using the Biotage SPE-DRY. Post lyophilization, custom exome bait (TWIST Biosciences) along with hybridization mastermix is added to the lyophilized pool prior to resuspension. Samples are incubated overnight. Library normalization and hybridization setup are performed on a Hamilton Starlet liquid handling platform, while target capture is performed on the Agilent Bravo automated platform. Post capture, a PCR is performed to amplify the capture material.
After post-capture enrichment, library pools are quantified using qPCR (automated assay on the Agilent Bravo), using a kit purchased from KAPA Biosystems with probes specific to the ends of the adapters. Based on qPCR quantification, pools are normalized using a Hamilton Starlet to 2 nM and sequenced using Illumina sequencing technology.

After library construction, hybridization and capture were performed using the relevant components of IDT’s XGen hybridization and wash kit and following the manufacturer’s suggested protocol, with several exceptions. A set of 12-plex pre-hybridization pools were created. Custom exome bait (TWIST Biosciences) along with hybridization mastermix was added to the lyophilized pre-hybridization pool prior to resuspension. Library normalization and hybridization setup were performed on a Hamilton Starlet liquid handling platform, while target capture was performed on the Agilent Bravo automated platform. Post capture, a PCR was performed to amplify the capture material. After post-capture enrichment, library pools were quantified using qPCR (automated assay on Agilent Bravo), using a kit purchased from KAPA Biosystems with probes specific to the ends of the adapters. Based on qPCR quantification, pools were normalized using a Hamilton Starlet to 2nM and sequenced using Illumina sequencing technology. The targeted panel bait set used in this study was designed at the Broad Institute to maximize pan-cancer utility and contains regions from 396 driver genes previously annotated in cancer literature.