Targeted Sequencing

Data Generation Protocols Data Analysis Protocols
CaCxIcon for HTMCP- Cervical Cancer Project CaCxIcon for HTMCP- Cervical Cancer Project

Data Generation Protocols

The data generation protocols for the HTMCP-Cervical Cancer project were acquired from the following manuscript.

Gagliardi A, Porter VL, Zong Z, et al. Analysis of Ugandan cervical carcinomas identifies human papillomavirus clade-specific epigenome and transcriptome landscapes. Nat Genet. 2020;52(8):800-810. (PMID: 32747824)

Genome library construction for custom capture

DNA (500 ng) was sonicated (Covaris) to 250-350 bp, purified using PCRClean DX magnetic beads (Aline Biosciences), end-repaired, phosphorylated and bead purified before A-tailing using a custom NEB Paired-End Sample Prep Premix Kit. Illumina sequencing adapters were ligated overnight at 16oC, bead purified and enriched with 6 cycles of PCR using indexed primers enabling library pooling and sequenced using paired-end 125 base reads in a single flowcell lane.

Custom capture validation of SNVs

Out of the cohort of 212 cervical cancer patients, 118 comprised the discovery cohort and 89 comprised the extension cohort. From the 89 extension cohort, targeted sequencing was performed in two phases. The first phase was to capture 2,735 selected genes, and the second phase was to capture the KMT2D gene and non-coding hotspots.

DNA libraries from the 89 patients were pooled prior to hybridization capture of 2,735 target genes using SureSelect XT custom probes (Agilent) and RNA probes at 65°C for 24 hours. Streptavidin-coated magnetic beads (Dynal, MyOne) were used for custom capture, followed by purification on MinElute columns (Qiagen) and enrichment with 10 PCR cycles using primers that maintain library-specific indices. Pooled libraries were sequenced generating 125bp paired-end reads. To capture the KMT2D gene and non-coding hotspots, 544 120bp xGen Lockdown probes were designed and synthesized (Integrated DNA Technologies) for targeted capture sequencing.

Experimental protocols

To request more information or approval regarding the following protocols, please contact BC Cancer at labqa@bcgsc.ca

Hybridization Capture of Illumina Libraries using xGEN Lockdown Probes

Multiplex Agilent Whole Exome or Target Capture

Data Generation Protocols

The data generation protocols for the HTMCP-Cervical Cancer project were acquired from the following manuscript.

Gagliardi A, Porter VL, Zong Z, et al. Analysis of Ugandan cervical carcinomas identifies human papillomavirus clade-specific epigenome and transcriptome landscapes. Nat Genet. 2020;52(8):800-810. (PMID: 32747824)

Somatic alteration detection

Tumor and normal sequencing reads were aligned to the human reference genome (hg19) using BWA-MEM v0.7.6a). Read duplicates were marked using sambamba (v0.5.5). Somatic single nucleotide variants (SNVs) were identified using Strelka (v1.0.6). A panel of 2,735 genes including mutated oncogenes, tumor suppressors, epigenetic modifiers, splicing factors, or other genes recurrently mutated (≥3 cases) in this cohort, were selected for targeting sequencing in the extended cohort. The coding mutation rate was reported for each tumor as the number of coding SNVs (low, moderate or high SNPeff annotation) per Mb.

Mutation signatures and HRD score

SNVs were categorized into 96 mutation classes based on 6 variant types and 16 trinucleotide contexts. For each sample, values of the 96 classes were used to compute a non-negative least squares deconvolution based on 30 previously described mutational signatures (COSMIC). The APOBEC signature reported for each sample is the max exposure value of signature 2 or 13.

HRD scores were computed using the R package HRDtools (v0.0.0.9), as the arithmetic sum of loss of heterozygosity (LOH), TAI, and LST scores, determined based on published guidelines.

Last updated: August 07, 2020