Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing
Articolo
Data di Pubblicazione:
2022
Citazione:
Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing / F.P. Fabrizio, S. Castellana, F. Centra, A. Sparaneo, M. Mastroianno, T. Mazza, M. Coco, D. Trombetta, N. Cingolani, A. Centonza, P. Graziano, E. Maiello, V.M. Fazio, L.A. Muscarella. - In: FRONTIERS IN ONCOLOGY. - ISSN 2234-943X. - 12:(2022), pp. 968804.1-968804.14. [10.3389/fonc.2022.968804]
Abstract:
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 aberrant methylation events occur simultaneously in a group of genes, thus providing a growth advantage to the cell in promoting cell differentiation and neoplastic transformation. Due to this reason, methylation profiles have been suggested as promising cancer biomarkers. Here, we designed and performed a first step of validation of a novel targeted next generation sequencing (NGS) panel for methylation analysis, which can simultaneously evaluate the methylation levels at CpG sites of multiple cancer-related genes. The OPERA_MET-A methylation panel was designed using the Ion AmpliSeq™ technology to amplify 155 regions with 125-175 bp mean length and covers a total of 1107 CpGs of 18 cancer-related genes. The performance of the panel was assessed by running commercially available fully methylated and unmethylated control human genomic DNA (gDNA) samples and a variable mixture of them. The libraries were run on Ion Torrent platform and the sequencing output was analyzed using the “methylation_analysis” plugin. DNA methylation calls on both Watson (W) and Crick (C) strands and methylated:unmethylated ratio for each CpG site were obtained. Cell lines, fresh frozen and formalin-fixed paraffin-embedded (FFPE) lung cancer tissues were tested. The OPERA_MET-A panel allows to run a minimum of 6 samples/530 chip to reach an observed mean target depth ≥2,500X (W and C strands) and an average number of mapped reads >750,000/sample. The conversion efficiency, determined by spiking-in unmethylated Lambda DNA into each sample before the bisulfite conversion process, was >97% for all samples. The observed percentage of global methylation for all CpGs was >95% and <5% for fully methylated and unmethylated gDNA samples, respectively, and the observed results for the variable mixtures were in agreement with what was expected. Methylation-specific NGS analysis represents a feasible method for a fast and multiplexed screening of cancer patients by a high-throughput approach. Moreover, it offers the opportunity to construct a more robust algorithm for disease prediction in cancer patients having a low quantity of biological material available.
Tipologia IRIS:
01 - Articolo su periodico
Keywords:
biomarker; cancer; driver gene; methylation; next generation sequencing
Elenco autori:
F.P. Fabrizio, S. Castellana, F. Centra, A. Sparaneo, M. Mastroianno, T. Mazza, M. Coco, D. Trombetta, N. Cingolani, A. Centonza, P. Graziano, E. Maiello, V.M. Fazio, L.A. Muscarella
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