Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing

Comprehensive evaluation of m⁶dA detection based on SMRT-seq data. (A,B) Sensitivity-FDR curves at different levels of per strand SMRT-seq coverage (A) and fraction of methylated A sites in the genome (B). Curves are estimated based on either P-value or IPD ratio; both are shown. FDR estimation is based on the coverage-matched native (Escherichia coli with m⁶dA at GATC sites; Methods) and WGA samples. (C) FDRs estimated for different combinations of per strand SMRT-seq coverage and fraction of m⁶dA sites, f(m⁶dA/A), in the genome. FDR estimation is based on the coverage-matched native and WGA samples (Methods) at an IPD ratio of four. (D) Motif specific methylation detection leads to more reliable m⁶dA calls with lower FDRs. (E) Distribution of P-values (−log₁₀) and IPD ratios of m⁶dA events (red) and nonmethylated A's (black) from 11 well-characterized bacterial m⁶dA methylomes. (F) Enrichment score for motifs with different fractions of motif sites methylated across the genome f_m(m⁶dA/A), estimated based on P-value (−log₁₀; left) and IPD ratio (right). SMRT-seq data from 11 bacterial species/strains with well-characterized m⁶dA methylomes are used for this simulation analysis. (G) Schematic illustrating single-molecule-level analysis for the estimation of partial methylation. A single molecule (two DNA strands and two adapters) and the subreads that are produced from the top strand of this molecule in SMRT-seq (top). For a given genomic position, when non-single-molecule analysis is performed, IPD ratios for the methylated and nonmethylated subreads follow two exponential distributions (red and black curves in the second panel). In contrast, when single-molecule analysis was performed, IPD ratios across all molecules follow two normal distributions with smaller variance over increasing coverage per molecule strand (third and fourth panels). (H) Estimation of partial methylation f_l(m⁶dA/A) by aggregate analysis (left) and single-molecule-level analysis (right). x-axis indicates background truth f_l based on simulation; y-axis, estimated f_l; and dots, 4359 A's with known fraction of m⁶dA methylation based on subsampling from a well-characterized E. coli m⁶dA methylome. (I,J) Distribution of IPD ratios for partially methylated m⁶dA sites and nonmethylated A's based on aggregate analysis (I) and single-molecule level analysis (J). The inset provides an enlarged view. The motif enrichment score for the same, known methylation motif GATC significantly differs between the two types of analyses (1.3 in aggregated analysis vs. 25 in single-molecule analysis).