EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

Abstract

Bisulfite sequencing is widely used to detect 5mC and 5hmC at single base resolution. It is the most accepted method for detecting these cytosine modifications, but it does have significant drawbacks. DNA is frequently damaged resulting in fragmentation, loss of DNA and inherent biases introduced to sequencing data. To overcome this, we developed a new method called Enzymatic Methyl-seq (EMseq). This method relies on two sets of enzymatic reactions. In the first reaction, TET2 and T4-bGT convert 5mC and 5hmC into substrates that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines converting them to uracils. The protection of 5mC and 5hmC permits the discrimination of cytosines from 5mC and 5hmC. Over a range of DNA inputs, the overall fraction of 5mC and 5hmC in EM-seq libraries was similar to bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite converted libraries in all specificmeasures examined including coverage, duplication, sensitivity and nucleotide composition. EM-seq libraries displayed even GC distribution, improved correlation across input amounts, increased numbers of CpGs confidently assessed within genomic features, and improved the accuracy of cytosine methylation calls in other contexts. Bisulfite sequencing is known to severely damage DNA thus making library construction for lower DNA input very difficult. We show that EM-seq can be used to make libraries using as little as 100 pg of DNA. These libraries maintain all of the previously described advantages over bisulfite sequencing thus opening new avenues for research and clinical applications. Even with challenging input material, EM-seq provides a method to detect methylation state more reliably than WBGS.[7]. Sequencing distinguishes cytosines from these modified forms as they are read as thymines and cytosines respectively [8]. Despite its widespread use amongst epigenetic researchers, bisulfite sequencing also has significant drawbacks. It requires extreme temperatures and pH which causes depyrimidination of DNA resulting in DNA degradation [9]. Furthermore, cytosines are damaged disproportionately compared to 5mC or 5hmC. As a result, sequencing libraries made from converted DNA have an unbalanced nucleotide composition. All of these issues taken together result in libraries with reduced mapping rates and skewed GC bias plots, with a general under-representation of G-and Ccontaining dinucleotides and over-representation of AA-, AT-and TA-containing dinucleotides, when compared to a non-converted genome [10]. Therefore, the damaged libraries do not adequately cover the genome, and can include many gaps with little or no coverage. Increasing the sequencing depth of these libraries may recover some missing information, but at steep sequencing costs.These bisulfite library limitations have driven the development of new approaches for mapping 5mC and 5hmC, in combination or independently, for epigenome analysis. The methylation dependent restriction enzymes (MDRE), MspJI ...

Comprehensive evaluation of targeted multiplex bisulphite PCR sequencing for validation of DNA methylation biomarker panels

“…This is not ideal for working with clinically derived DNA which can already be degraded and low yield. Recently, new techniques have been developed to address this problem, such as TET-assisted pyridine borane sequencing (TAPS) [43] and enzymatic methylsequencing (EM-seq) [44]. These methods both rely on TET enzymatic reactions to deaminate methylated cytosines.…”

Mapping the epigenetic modifications of DNA and RNA

“…EM-Seq showed higher mapping efficiency and more uniform GC coverage than BS-Seq. However, conversion of all unmodified C to U by EM-Seq would still cause low complexicity problem in the sequencing library and lower DNA input such as 100 pg resulted in PCR duplicate rate as high as 84.5% while only 10.8% of the reads were usable (Vaisvila et al, 2019 ).…”

Pervasive non-CpG methylation at zebrafish mosaic satellite repeats

“…Bisulfite-converted (WGBS) and APOBEC-converted (EM-seq) [14] sequence reads were trimmed with Trimmomatic (ILLUMINACLIP:TruSeq3-SE.fa:2:30:10 SLIDINGWINDOW:5:20 LEADING:3 TRAILING:3 MINLEN:20) [42], and mapped using WALT (-m 5 -t 20 -N 10000000) [43] onto the bisulfite-converted GRCz11 reference (UCSC) containing λ (WGBS and EM-seq) and pUC19 sequences (EM-seq) added as separate chromosomes. The resulting alignments in BAM format were processed with CGmapTools [43,44] (convert bam2cgmap) to obtain methylation calls.…”

“…Even under such stringent mapping conditions we observe substantial coverage of MOSAT_DR repeats and confirm the strand bias associated with TGCT methylation (Additional File 1: Figure S2). Additionally, to confirm that this form of methylation is not the result of a sequence-specific bias of bisulfite conversion [13], we generated enzymatic methylation sequencing (EM-seq) base-resolution libraries of two biological replicates of 24hpf embryos [14 ](Additional File 1: Table S1). The top methylated MOSAT motifs identified in either EM-seq or WGBS showed proportionate methylation levels when compared to the orthogonal approach (Fig.…”