Use of Synthetic Reference Sequence Spike-In Controls to Increase Reliability of NGS Testing for Actionable Mutations in Circulating Tumor DNA

Use of Synthetic Reference Sequence Spike-In Controls to Increase Reliability of NGS Testing for Actionable Mutations in Circulating Tumor DNA

Abstract
Introduction: Recent development of Next Generation Sequencing (NGS) methods that measure the methylome in circulating tumor DNA have potential to enable earlier detection of cancer and more sensitive measurement of minimal residual disease. Advances include methods that measure cancer-type specific methylated regions to enable determination of site of origin and methods that measure hundreds to thousands of methylated regions and thereby enable earlier detection by overcoming stochastic sampling associated with measurement of a small number of targets. Wide-spread adoption of these methods for clinical diagnostic applications will be facilitated through improved quality controls that reduce false positive and false negative results and increase reliability. The purpose of this study was to use recently developed DNA methylation test materials developed by the National Institute for Standards and Technology (NIST) to assess performance of the SNAQ-SEQ™ method as a quality control to establish limit of detection (LOD) for measurement of methylation at each targeted CpG site.
Methods: NIST test materials were provided through an inter-laboratory pilot testing program. These materials were formulated by mixing DNA from NA24385 (Coriell Institute for Medical Research) in the “native” state of DNA with in vitro methylated genomic DNA (methylated NA24385) at fractions of 0%, 5%, 20%, 50%, and 100% (Samples A-E). The genome-wide DNA methylation pattern was analyzed by reduced representation bisulfite sequencing (RRBS). The Accugenomics, Inc. SNAQ-SEQ™ Mix4 kit was used in these studies. Synthetic internal standards (IS) included in this kit, and PCR primers designed for analysis of post-bisulfite treated DNA were used to assess LOD for measurement of methylation at each of 14 CpG sites in the targeted region of the SOX2 gene. Following mixture of Mix4 IS with each of the NIST test materials, each mixture was subjected to PCR-amplicon library preparation, then sequenced on Illumina MiSeq.
Results: Based on RRBS conducted at NIST, average methylation across CpG in the SOX2 gene was 11.6% in native NA24385 DNA (Sample A), and increased as expected to a maximum of 98% (Sample E). In our analysis of the particular SOX2 region targeted by the SNAQ-SEQ™ Mix4 kit, average methylation (preserved C nucleotide) was 16.4% (0.5%-50%) in native NA24385 DNA. In contrast in IS corresponding to the targeted SOX2 region the average preserved C was 0.32% (0.03%-0.5%).
Conclusions: Use of synthetic internal standards enabled determination of LOD for each individual methylated site, controlling for all known sources of interference and technical error, including inefficient bisulfite conversion, nucleotide substitution error, and alignment error.