WES of homo sapiens: TNBC Tumor
Paraffin from 2.5uM-thick tumor sample sections (8-10) was removed with xylene, followed by ethanol washes, and then DNA extraction was performed using High Pure FFPET DNA Isolation Kit (Roche, Ref 06650767001) following manufacturer instructions. Briefly, deparaffinized tissue pellet is incubated with lysis buffer and proteinase K during 60 min at 56C. After inactivation of enzyme at 90C, tissue lysates were placed into high pure filter tubes, previously adding DNA binding buffer and isopropanol. After washing column with buffers I and II, DNA was eluted in a fresh 1.5-ml reaction tube. DNA was then quantified using the Qubit 1X dsDNA HS Assay Kit (ThermoFisher Scientific) in the Qubit 4 Florometer (ThermoFisher Scientific). DNA integrity evaluation was performed using the Genomic DNA Assay kit (Revvity) on the LabChip GX Touch (Revvity). Libraries were prepared using the Illumina DNA Prep with Enrichment v.2.5 kit as recommended by the manufacturer. A set of 12-plex pre-hybridization pools were created and included for their hybridization with the Twist Bioscience for Illumina Exome 2.5 Panel (Illumina). Library pools were quantified using Qubit 1X dsDNA HS Assay Kit (ThermoFisher Scientific) following the manufacturers suggested protocol in the Qubit 4 Florometer (ThermoFisher Scientific). The final library size were determined using High Sensitivity DNA Assay (Revvity) on the LabChip GX Touch (Revvity). Libraries were sequenced using Illuimina NovaSeq X Plus sequencing system (Illumina) at a loading concentration of 150pM in a 10B Flowcell (2x150bp). Illumina BCL Convert software were used for generating fastq files.