Abstract 3750: Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology
Authors: Fabrizio Federico Pio , Castellana Stefano , Sparaneo Angelo , Mazza Tommaso , Centra Flavia , Trombetta Domenico , Fazio Vito Michele , Muscarella Lucia Anna 
Published: June 2022

Abstract
Background: DNA methylation is the most recognized epigenetic mark that leads to a massive distortion in cancer cells. It has been observed that a large number of DNA hypermethylation events co-occur in groups of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Here we designed and performed a first step of validation of a novel next-generation sequencing (NGS) targeted methylation panel that can simultaneously evaluate the methylation levels of multiple cancer-related genes.
Methods: The WG00446_MET_A AmpliSeq™ Methylation Panel for Cancer Research was designed using the Ion AmpliSeq technology to amplify 152 regions with an amplicon size range of 125-175bp, that covers a total of 1107 promoter and intragenic CpGs of 18 cancer-related genes. The performance of the panel was assessed by running in duplicate commercially available fully methylated and unmethylated control human gDNA samples and a variable mixture of them to simulate 10%, 25%, 75%, and 90% of global methylation. The libraries (manual preparation using the Ion AmpliSeq Plus Library Kit) were loaded on 530 chips and run on the GeneStudio S5 Sequencer (Thermo Fisher Scientific). The sequencing output was analyzed using the methylation_analysis plugin, available on Ion Torrent Suite v5.16 (Thermo Fisher Scientific).
Results: The WG00446_MET_A panel allowed to run 6 samples per 530 chips to reach an observed mean target depth &gt;2,000X (observed reads on-targets: &gt;80%; average number of mapped reads: &gt;450,000/sample). The conversion efficiency, determined by spiking-in Unmethylated Lambda DNA into each sample before the bisulfite conversion process, was &gt;97% for all samples. The observed % of global methylation for all CpGs were &gt;98% and &lt;2% for fully methylated and unmethylated gDNA samples, respectively. The observed results for the variable mixtures were in agreement with what expected.
Conclusions: The methylation-specific NGS analysis using the WG00446_MET_A panel represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. It will offer the opportunity to design a more robust algorithm for disease prediction for those cancer patients with a low quantity of biological material available. The orthogonal testing on DNA extracted by FFPE and frozen samples is ongoing with a subset of amplicons from the WG00446_MET_A panel.
Citation Format: Federico Pio Fabrizio, Stefano Castellana, Angelo Sparaneo, Tommaso Mazza, Flavia Centra, Domenico Trombetta, Vito Michele Fazio, Lucia Anna Muscarella. Design and experimental validation of WG00446_MET_A methylation next generation sequencing panel using ion AmpliSeq technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3750.