The assessment by NGS of FFPE derived tumor DNA using an ovarian cancer and a custom solid tumor hybridization-based enrichment panel approach

Introduction

• One of the challenges in cancer research is the high level of genetic complexity and tumor heterogeneity. 
• Research that generates detailed information about the genetic profile of each individual tumor will further our understanding and may be used in the future to guide treatment strategies¹. 
• Next Generation Sequencing (NGS) has enabled the simultaneous study of multiple mutations in high-penetrance cancer predisposition genes. However, tissue biopsies are typically archived as formalin-fixed, paraffin embedded (FFPE) blocks which can significantly compromise the quality and amount of nucleic acids available for genomics research.

To overcome these issues, we have used the SureSeq™ FFPE DNA Repair Mix*, in combination with a hybridization-based NGS custom enrichment panel, the SureSeq Ovarian Cancer Panel (Figure 1) to identify somatic variation in key DNA repair genes associated with ovarian cancer.

Hybridization-based enrichment generates highly uniform coverage of key targets

To confidently call low level variants, NGS reads need to be evenly distributed across all regions of interest. Uniformity of coverage is a useful value with which to compare this distribution and can be expressed as the percentage of target bases that have greater than 20% of the mean coverage. As reported extensively in the literature^1-3, the uniformity of coverage from capture-based approaches such as SureSeq consistently outperform those enriched using amplicon-based methods (Figure 2). Furthermore, in our sample set we found the high levels of uniformity are maintained when starting with ~250 ng DNA (light blue bars).

The uniformity of the coverage for most samples is greater than 99% of bases covered at 20% of the mean, ensuring that all bases within the panel can be assessed.

Somatic variants can be confidently detected in type II ovarian cancer research samples

The superior uniformity of coverage enables reliable identification of somatic single nucleotide variants (SNVs) and indels in solid tumor samples. Figure 3 illustrates some examples of somatic deletions (panel A) and SNVs (panel B) that have been found in exon 6 of TP53 from FFPE blocks of type II EOC samples.

Formalin-damage in DNA can be reduced through use of FFPE DNA repair mix

We found pre-treatment with the SureSeq FFPE DNA Repair Mix improves the mean target coverage of formalin comprised samples (Horizon Diagnostics), thereby increasing the flexibility of the assay (Figure 4). Use of the repair mix also enables a reduced DNA input down to 50 ng whilst maintaining a good depth of coverage (Figure 5).

Accurate detection of variants from reference standards

All samples had 100% concordance for 20 reported variants with 97.5% having allele frequencies within 5% of the expected value (Table 1).