Abstract 158: Custom primer design pipeline and analysis workflow for targeted methylation sequencing using NGS Ion AmpliSeq technology

Luo, Zunping; Pickle, Loni; Hatch, Andrew G.; Ewing, Aren; Hyland, Fiona; Berman, David M.; Patel, Palak; Andersen, Mark

doi:10.1158/1538-7445.am2020-158

Cited by 3 publications

(5 citation statements)

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“…Anyway, a high intra-samples variability was observed among gDNA from all samples type ( Supplemental Table 3 ). The mean number of read per amplicon was 2862 ± 304 for W strand (ranging from 1035 to 5875), and 3081 ± 345 for C strand (ranging from 333 to 6433), in line with the data previously reported by Luo et al for the Ion AmpliSeq™ Methylation Panel for Cancer Research ( 15 ).…”

Section: Resultssupporting

confidence: 90%

“…This panel allows to obtain libraries starting from gDNA extracted from different matrices, such as cell lines, OCT embedded frozen and FFPE tissues. Considering the recently published data on NGS for methylation analysis using the gene-targeted AmpliSeq technology, very promising and comparable results to those already been published were obtained ( 15 ) in terms of mean target depth ≥2,500X (W and C strand), average number of mapped reads >750,000/sample and concordance results between expected and observed % of global methylation for all CpGs. More specifically, the OPERA_MET-A panel primer design pipeline include more amplicons (155 amplicons) than those generated by Ion Ampliseq™ Methylation Panel for Cancer Research (40 amplicons) that covers 18 non-overlapping genes and can be used to analyze gDNA from cell lines, human FFPE and OCT tissues to reach a comparable performance in terms of time consuming, observed/expected methylation rate concordance and average mapped reads.…”

Section: Discussionsupporting

confidence: 73%

“…The methylation analysis was performed using the previously described outline ( 14 , 15 ) and the Ion Torrent Suite™ Software (version 5.10.1) running on the Torrent Server (Thermo Fisher Scientific) was used to process the sequencing data. The “methylation_analysis” Torrent Suite plugin (Thermo Fisher Scientific) was used to analyze the sequencing output of the OPERA_MET-A panel and annotate the percentage of each targeted CpG site.…”

Section: Methodsmentioning

confidence: 99%

“…This analysis plugin performs sequencing read alignment onto the W and C strands of the GRCh37/hg19 reference genome and then assesses the methylation status in a strand-specific manner. Reports and text files were generated for each amplicon, containing the number of methylated and unmethylated reads as well as the percentage of methylation per amplicon in relation to the targeted region/CpG sites ( 14 , 15 ). A summary report was created for each sample that includes the barcode name, the assigned sample name, the total number of reads that cover the target CpGs, and the percentage of methylated reads.…”

Section: Methodsmentioning

confidence: 99%

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Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing

et al. 2022

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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.

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Section: Resultssupporting

confidence: 90%

Section: Discussionsupporting

confidence: 73%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing

et al. 2022

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“…48 Such kit-based targeted next-generation sequencingebased assays have excellent quality control protocols and can incorporate DNA methylation analysis within a single test. 49 These tests have also been widely adopted in many hospitals and should have similar costs to uncomplicated prostate needle biopsies.…”

Section: Discussionmentioning

confidence: 99%

Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance

et al. 2023

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Purpose: Latent grade group 2 prostate cancer can impact the performance of active surveillance protocols. To date, molecular biomarkers for active surveillance have relied solely on RNA or protein. We trained and independently validated multimodal (mRNA abundance, DNA methylation, and/or DNA copy number) biomarkers that more accurately separate grade group 1 from grade group 2 cancers. Materials and Methods: Low-and intermediate-risk prostate cancer patients were assigned to training (n[333) and validation (n[202) cohorts. We profiled the abundance of 342 mRNAs, 100 DNA copy number alteration loci, and 14 hypermethylation sites at 2 locations per tumor. Using the training cohort with crossvalidation, we evaluated methods for training classifiers of pathological grade group 2 in centrally reviewed radical prostatectomies. We trained 2 distinct classifiers, PRONTO-e and PRONTO-m, and validated them in an independent radical prostatectomy cohort.

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Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks

Pośpiech,

Pisarek,

Rudnicka

et al. 2023

Clin Epigenet

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Background DNA methylation analysis has proven to be a powerful tool for age assessment. However, the implementation of epigenetic age prediction in diagnostics or routine forensic casework requires appropriate laboratory methods. In this study, we aimed to compare the performance of large-scale DNA methylation analysis protocols that show promise in terms of accuracy, throughput, multiplexing capacity, and high sensitivity. Results The protocols were designed to target a predefined panel of 161 genomic CG/CA sites from four known estimators of epigenetic age-related parameters, optimized and validated using artificially methylated controls or blood samples. We successfully targeted 96% of these loci using two enrichment protocols: Ion AmpliSeq™, an amplicon-based method integrated with Ion Torrent S5, and SureSelectXT Methyl-Seq, a hybridization-based method followed by MiSeq FGx sequencing. Both protocols demonstrated high accuracy and robustness. Although hybridization assays have greater multiplexing capabilities, the best overall performance was observed for the amplicon-based protocol with the lowest variability in DNA methylation at 25 ng of starting DNA, mean observed marker coverage of ~ 6.7 k reads, and accuracy of methylation quantification with a mean absolute difference between observed and expected methylation beta value of 0.054. The Ion AmpliSeq method correlated strongly with genome-scale EPIC microarray data (R = 0.91) and showed superiority in terms of methylation measurement accuracy. Method-to-method bias was accounted for by the use of linear transformation, which provided a highly accurate prediction of calendar age with a mean absolute error of less than 5 years for the VISAGE and Hannum age clocks used. The pace of aging (PoAm) and the mortality risk score (MRS) estimators included in our panel represent next-generation clocks, were found to have low to moderate correlations with the VISAGE and Hannum models (R < 0.75), and thus may capture different aspects of epigenetic aging. Conclusions We propose a laboratory tool that allows the quantification of DNA methylation in cytosines underlying four different clocks, thus providing broad information on epigenetic aging while maintaining a reasonable number of CpG markers, opening the way to a wide range of applications in forensics, medicine, and healthcare.

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Abstract 158: Custom primer design pipeline and analysis workflow for targeted methylation sequencing using NGS Ion AmpliSeq technology

Cited by 3 publications

References 0 publications

Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing

Design and experimental validation of OPERA_MET-A panel for deep methylation analysis by next generation sequencing

Multimodal Biomarkers That Predict the Presence of Gleason Pattern 4: Potential Impact for Active Surveillance

Introduction of a multiplex amplicon sequencing assay to quantify DNA methylation in target cytosine markers underlying four selected epigenetic clocks

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