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Abstract Introduction: DNA methylation plays a crucial role in development and progression of cancer. Identification of DNA methylation profiles in cancer is indispensable for early detection. To address this research need, Ion AmpliSeqTM offers targeted next-generation sequencing (NGS) methylation community amplicon panels for DNA methylation profiling in cancer-associated genes. However, the process of designing custom methylation amplicon panels using the Ion AmpliSeq primer design pipeline is intricate, labor-intensive, and time-consuming. Here, we developed a one-step, fully automated, faster, and reproducible nextflow-based primer design pipeline (MetFlow) for development of custom Ion AmpliSeqTM methylation panels. MetFlow streamlines the development of custom methylation panels for cancer-related genes, and thus significantly reduces the time and effort traditionally required for amplicon design. Methods: The fully automated MetFlow pipeline is implemented using Nextflow. MetFlow necessitate an input configuration file for the execution of methylation Ion AmpliSeqTM designs. The configuration file specifies the essential parameters, including target CpG input, reference genome, pool number, and amplicon lengths, which are required for designing the amplicons. The MetFlow pipeline designs candidate amplicons for CpG targets on both Watson and Crick strands, facilitating the subsequent creation of files required for the generation of a methylation panel. Results: The fully automated MetFlow pipeline has been used for designing the customized Ion AmpliSeqTM methylation amplicon panel (s) for cancer and age-related genes. The amplicons lengths in these AmpliSeqTM methylation panels were ranged from 125 bp to 175 bp for formalin-fixed, paraffin-embedded (FFPE) research samples. These customized methylation panels were run on the Ion GeneStudioTM S5 sequencer followed by performance analysis using Ion TorrentTM Suite. The published data for these custom amplicon panels suggest that the Ion AmpliSeqTM has sufficient read depth and mapping efficiency, higher sensitivity and accuracy for methylation measurement, and strong correlation (r=0. 993) for methylation profiling among sample replicates. Conclusion: We present MetFlow, an advanced automated primer design pipeline for designing the custom Ion AmpliSeqTM methylation amplicon panels tailored to targeted NGS applications in cancer research. The custom designed Ion AmpliSeqTM methylation panels exhibited sufficient read depth, higher sensitivity and accuracy, and reproducibility. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Renesh Bedre, Drishti Kaul, Anupma Sharma, Andrew Hatch, Loni Pickle, Na Li, Fiona Hyland. MetFlow: Automated nextflow pipeline for Ion AmpliSeq methylation assay designs abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3489.
Bedre et al. (Fri,) studied this question.