PANDA: Read-Level Phased Analysis of DNA Amplicons for Methylation Studies

Abstract DNA methylation analysis using bisulfite sequencing is widely used to investigate epigenetic regulation at single-base resolution; however, conventional analysis workflows primarily rely on site-wise averaging, which obscures contiguous methylation patterns encoded within individual DNA molecules and limits interpretation of epiallelic heterogeneity in targeted amplicon studies. Here, we present PANDA (Phased ANalysis of DNA Amplicons), an end-to-end graphical pipeline that restores contiguous single-molecule methylation patterns by linking unmerged paired-end reads to reconstruct epiallelic patterns across unsequenced regions. PANDA supports both Sanger and next-generation sequencing inputs, providing a unified workflow for alignment, read-level methylation calling, phased visualization, and quantification of within-sample methylation heterogeneity. Using synthetic benchmarking datasets, we demonstrated that in silico motif filtering isolates specific target reads, enabling the accurate detection of allele-specific methylation and loss of imprinting. Furthermore, the re-analysis of primate placentae datasets confirmed that long-range phasing across unsequenced regions successfully restored the original epiallelic architectures. PANDA establishes a robust, practical approach to single-molecule epigenomic profiling using targeted bisulfite amplicon sequencing. Competing Interest Statement The authors have declared no competing interest. Abbreviations - ASM - allele-specific methylation - DMR - differentially methylated region - FDR - false discovery rate - LOI - loss of imprinting - NGS - next-generation sequencing - PDR - proportion of discordant reads - QC - quality control - qFDRP - quantitative fraction of discordant read pairs - RMSE - root mean square error - SD - standard deviation - SNP - single-nucleotide polymorphism - TF - transcription factor