Dragen germline pipeline

WGS data was processed using the Illumina DRAGEN Germline pipeline v3.8.4 with default settings. Due to the volume of data, each lane was processed individually and the resulting variant call format (vcf) files were merged for variant analysis. WES data was processed using the DRAGEN Enrichment pipeline v3.8.4 with default settings. Additionally, each pipeline was used to process the Mock and Edited samples as “tumor/normal” and “normal/tumor” pairs. Known systematic noise filters were applied to all called variants. Data visualization was done in Prism (version 9) and RStudio (Version 1.2.5033).

NGS data were analyzed by the Illumina Dragen Germline pipeline (Dragen version 4.0.3, Illumina) where both sequence variants and copy number alterations were assessed. GRCh37 genome build and NM_000546.5 (MANE Select transcript) were used as reference sequence. Variants were reclassified following the guidelines of the ClinGen TP53 Expert Panel specifications^{14 (link),15 (link)}. TP53 variants were also cross-checked in the NCI TP53 database, NCBI ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), Clinical Genome Resource (https://clinicalgenome.org/), Varsome (https://varsome.com/) and Franklin (https://franklin.genoox.com/clinical-db/home) databases. Variant interpretation and cross-referencing in different databases were done between 2 and 21 December 2022, therefore the abovementioned database data are presented accordingly.

All animals were maintained according to Louisiana State University Institutional Animal Care and Use Committee (IACUC) approved standards. Adult female (8-12-week-old) BPH/5 mice from an in-house colony (Louisiana State University), and BPH/2 and C57Bl/6 female mice purchased from Jackson Laboratory were used. All animals were maintained according to Louisiana State University Institutional Animal Care and Use Committee (IACUC) approved standards. Total Genomic DNA was isolated from tail snips of 3 mice belonging to each strain (BPH/5, BPH/2 and C57Bl/6) using DNeasy Blood & Tissue Kit from Qiagen, and sequencing libraries were generated using the Nextera DNA Flex library kit (Illumina, San Diego, CA). All samples were sequenced with paired end 150bp reads on the Illumina NovaSeq (Illumina, San Diego, USA). Raw reads were mapped to the mm10 reference genome using the Illumina DRAGEN Germline Pipeline (v3.2.8). Each mouse replicate variants were merged with bcftools (v1.10) and unique/common variants were pulled for cross comparisons with vcftools (v0.1.17). Variants were annotated for putative impacts with Ensembl Variant Effect Predictor (v101.0) to the mm10 genome. Gene ontology (GO) enrichment was performed via PANTHER to identify significantly enriched biological processes (FDR p<0.05) and those ontologies present on chromosome 8 and X.