Assessment of the performance of a hybridisation-based NGS enrichment panel with as little as 10 ng of severely formalin-compromised DNA

Jacqueline Chan, Juliette Forster, Aysel Heckel, Venu Pullabhatla, Dave Cook, Graham Speight
Oxford Gene Technology (OGT)

Introduction

Formalin-fixed, paraffin-embedded (FFPE) storage is a standard method for archiving samples from solid tumours. It ensures the preservation of the ultrastructure of tissues and prevents degradation through formation of chemical links between macromolecules, for example between and within DNA molecules. FFPE samples contain a wealth of information which can be used to study cancer development and progression. Next generation sequencing (NGS) offers the capability of unlocking this information through the simultaneous study of multiple types of mutations in cancer-associated genes for a number of applications¹. However, formalin treatment can significantly compromise the quality and amount of nucleic acids available for genomics research. As such it is technically challenging to examine the true genetic complexity present in a sample.

In this study DNA reference standards with different levels of formalin-induced damage were hybridised and sequenced with a SureSeq^TM custom NGS panel in conjunction with the SureSeq FFPE DNA Repair Mix*. We assessed the impact of the repair mix on three levels of formalin compromised DNA (fcDNA) - ‘mild’, ‘moderate’ and ‘severe’, at 4 different DNA input amounts down to 10 ng. We then compared the uniformity and coverage of the enriched targets. We also assessed the concordance to the allele frequencies of the variants in the reference standards.

Study design

We tested three reference standards of fcDNA (provided by Horizon Discovery^†) with ‘mild’, ‘moderate’ and ‘severe’ damage². The samples were investigated in duplicate before and after repair with SureSeq FFPE DNA Repair mix; the amounts of input DNA were 200, 100, 50 and 10 ng (Figure 1). All samples were sheared using a Covaris S220 focused-ultrasonicator and prepared using the SureSeq NGS Library Preparation Kit (cat. no. 500070).

Enrichment by hybridisation was completed with a SureSeq custom NGS 8.7 Kb custom hot-spot panel designed to target the variants present in the reference standards (see Table 1 for variants targeted). The subsequent post-capture libraries were sequenced on an Illumina MiSeq^® using a v2 300 cycles kit (cat. no. MS-102-2002). 16 samples were run on a MiSeq lane.

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