HRD score Analysis

Background

HRD Overview

Homologous recombination repair (HRR) is an important pathway for normal cells to repair DNA damage. The loss-of-function variants in BRCA1/BRCA2 may directly result in Homologous Recombination Deficiency (HRD). Meanwhile, the mutation occurred in PALB2, CDK12, RAD51 and other HRR-related genes, as well as other unknown reason, may also lead to HRD. Generally, the detectable genomic aberrations caused by HRD are called "Genomic Scars", including loss of heterozygosity (LOH), large scale state transition (LST) and telomeric allelic imbalance (TAI) ^[1-2]. These signatures may act as biomarkers for the state of DNA repair deficiency.

HRD and Poly ADP-ribose polymerase inhibitor (PARPi)

In normal cells, the HRR mechanism allows cells to repair DNA replication errors and survive. For tumor cells with HRD, PARPi suppresses DNA replication forks and DNA replication, leading to cell apoptosis, which achieves anti-tumor effect.

HRD Clinical significance of HRD detection

Screen potential benefits of platinum and PARPi for Patients
Detection of genetic disease, such as hereditary breast cancer-ovarian cancer syndrome, Lynch syndrome.

Solution

外显子捕获应用-流程图

Library Preparation

Blocking

Target Capture

Sequencing

NadPrep DNA Universal Library Preparation Kit (for Illumina®)

Illumina^® Systems

NadPrep DNA Universal Library Preparation Kit (for MGI)

NadPrep NanoBlockers

(for MGI)

MGISEQ / DNBSEQ™

Performance of HRR Panel

Capture Performance

Pic1. Capture performance of HRR Panel v1.0. DNA libraries of human genomic DNA, male (Promega, G1471) were prepared by using the NadPrep DNA Library Preparation Kit (for Illumina®), and captured by using HRR Panel v1.0. The A. mapping rate, on-target rate, coverage uniformity, and B. coverage consistency were exhibited, respectively.Sequencing platform is NovaSeq 6000 with PE150.

Variant Analysis

Pic2. Variant analysis by HRR Panel v1.0. DNA libraries of Onco gDNA (Genewell, GW-OGTM800) were prepared by using the NadPrep DNA Library Preparation Kit (for Illumina®), and captured by using HRR Panel v1.0. The variant analysis was performed by Vardict. Sequencing platform is NovaSeq 6000 with PE150.

Spike-in Capture Performance

Pic3. Spike-in capture performance of HRR Panel v1.0 DNA libraries were prepared by using the NadPrep DNA Library Preparation Kit (for Illumina®), and captured by using HRR Panel v1.0 and HiSNP Ultra Panel v1.0 with equimolar. The A. mapping rate, on-target rate, B. coverage uniformity and consistency were exhibited, respectively.Sample type: Human genomic DNA, male (Promega, G1471), Onco gDNA (Genewell, GW-OGTM800), and Onco SNV Wildtype gDNA (Genewell, GW-OGTM005). Sequencing platform is NovaSeq 6000 with PE150.

Capture Performance of HiSNP-series Panel on MGI and Illumina Platform

Human Genomic DNA

Pic 4. Capture performance of HiSNP-series Panel on MGI and Illumina Platform. DNA libraries of human genomic DNA, male (Promega, G1471) were prepared by using the NadPrep DNA Library Preparation Module (for MGI) and the NadPrep DNA Library Preparation Kit (for Illumina®), respectively. The A. mapping rate, on-target rate, B & C. coverage uniformity and consistency of HiSNP (HiSNP Panel v1.0) and HiSNP Ultra (HiSNP Ultra Panel v1.0) were exhibited, respectively.

HRD Standard

Pic 5. Capture performance of HiSNP Ultra Panel v1.0 on MGI and Illumina Platform. DNA libraries were prepared by using the NadPrep DNA Library Preparation Module (for MGI) and the NadPrep DNA Library Preparation Kit (for Illumina®), respectively. The mapping rate, on-target rate, coverage uniformity and consistency of HiSNP Ultra Panel v1.0 were exhibited on A-B. MGI platform and C-D. Illumina platform. GW3, GW-HRD-3 standards (Genewell); GW9, GW-HRD-9 standards (Genewell); GW12, GW-HRD-12 standards (Genewell). All standards are paired cell lines, including Normal (N) and Tumor (T). MGI platform: MGISEQ-2000 with PE100; Illumina platform: NovaSeq 6000 with PE150.

HRD score Analysis

Pic 6. HRD score analysis of HiSNP Ultra Panel v1.0 with HRD standards. DNA libraries were prepared using the NadPrep DNA Library Preparation Kit (for Illumina®). All the WGS, WES (Exome Plus Panel v2.0) and HiSNP Ultra can be applied for HRD score analysis. Compared with WES, the score evaluated by HiSNP Ultra is closer to WGS and the reference value. Note: Sample type: HRD standards (Genewell). The data of WGS were analyzed using TITAN v1.17.1. The data of WES and HiSNP Ultra were analyzed using scarHRD v1.0. The sequencing depth of WGS, WES and HiSNP Ultra were over 30×, 300× and 450×, respectively. Illumina platform: NovaSeq 6000 with PE150.

Factors of HRD score Analysis

Tumor Purityscore

Pic 7. The influence of tumor purity on HRD score analysis. Nine simulated samples with different mixture proportions (10% - 90% Tumor) were generated by reads from tumor and normal sequencing FASTQfiles. were simulated to analyze the interaction withscoreWhen the purity of the tumor samples is between 30% to 40%, the HRD score value is relatively stable and close to the reference value. Ploidy, purity and HRD score were calculated by scarHRD v1.0 software.GW3, GW-HRD-3 standards (Genewell); GW9, GW-HRD-9 standards (Genewell); GW12, GW-HRD-12 standards (Genewell).

Sequencing Depth Score

Pic 8. The influence of sequencing depth on HRD score analysis. The sequencing data were further downsampled to 250x, 200X, 150x, 100x and 50x to analyze the interaction with purity, ploidy and HRD score by using scarHRD v1.0 software. The HRD score value could be accurately analyzed with 100% pure tumor samples when the sequencing depth was as low as 50x. GW3, GW-HRD-3 standards (Genewell); GW9, GW-HRD-9 standards (Genewell);

Influence of sequencing depth on HRD score analysis

Pic 9. The influence of tumor purity and sequencing depth on HRD score analysis. The sequencing depth were downsampled from 450x to 250x, 200x, 150x, 100x and 50x on in silico simulated samples with tumor purity between 30% and 40%, respectively. The scarHRD v1.0 software was used to analyze the interaction with purity, ploidy and HRD score. GW3, GW-HRD-3 standards (Genewell); GW9, GW-HRD-9 standards (Genewell);

Summary

HRD Recommendations for HRD analysis

HiSNP Ultra Panel v1.0 is suitable for HRD score analysis, which is better than WES and closer to WGS and HRD standards reference value in HRD score assessment.
HRD score analysis requires >30% of tumor sample purity and >300x of sequencing depth at least.
The feasibility of clinical HRD assessment depends on the simultaneous use of specific panels.

Pic 7. The implementation scheme of HRD assessment with HiSNP-series Panel along with HRR Panel.

Reference

[1] Stewart R A, Pilié P G, Yap T A. Development of PARP and immune-checkpoint inhibitor combinations[J]. Cancer research, 2018, 78(24): 6717-6725.

[2] Watkins J A, Irshad S, Grigoriadis A, et al. Genomic scars as biomarkers of homologous recombination deficiency and drug response in breast and ovarian cancers[J]. Breast Cancer Research, 2014, 16(3): 1-11.