Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA

Abstract

Bisulfite sequencing detects 5mC and 5hmC at single-base resolution. However, bisulfite treatment damages DNA, which results in fragmentation, DNA loss, and biased sequencing data. To overcome these problems, enzymatic methyl-seq (EM-seq) was developed. This method detects 5mC and 5hmC using two sets of enzymatic reactions. In the first reaction, TET2 and T4-BGT convert 5mC and 5hmC into products that cannot be deaminated by APOBEC3A. In the second reaction, APOBEC3A deaminates unmodified cytosines by converting them to uracils. Therefore, these three enzymes enable the identification of 5mC and 5hmC. EM-seq libraries were compared with bisulfite-converted DNA, and each library type was ligated to Illumina adaptors before conversion. Libraries were made using NA12878 genomic DNA, cell-free DNA, and FFPE DNA over a range of DNA inputs. The 5mC and 5hmC detected in EM-seq libraries were similar to those of bisulfite libraries. However, libraries made using EM-seq outperformed bisulfite-converted libraries in all specific measures examined (coverage, duplication, sensitivity, etc.). EM-seq libraries displayed even GC distribution, better correlations across DNA inputs, increased numbers of CpGs within genomic features, and accuracy of cytosine methylation calls. EM-seq was effective using as little as 100 pg of DNA, and these libraries maintained the described advantages over bisulfite sequencing. EMseq library construction, using challenging samples and lower DNA inputs, opens new avenues for research and clinical applications.

Correcting Methylation Calls in Clinically Relevant Low-Mappability Regions

“…The whole genome bisulfite (WGBS) libraries were prepared using the Ultra II DNA library prep kit (NEB E7645) before being Bisulfite converted using the Zymo EZ DNA Methylation-Gold bisulfite conversion kit according to the protocol described in Vaisvila et al [38].…”

“…The EM-seq libraries were prepared using the NEBNext Ultra II DNA library prep kit (NEB E7645) (using the NEBNext EM-seq adapter), then EM-seq converted using TET2 and ABOBEC3A, as described in Vaisvila et al [38].…”

“…EM-seq vs. WGBS Due to the reduced DNA damage and longer possible read lengths that are found in in EM-seq libraries, we compared with WGBS sequence libraries produced using the same 3 sample masses, to see if there is a dierence between the two methods with regard to the number of miscalls. WGBS libraries have fewer overall Cs called [38] and therefor fewer miscalled Cs at all input masses than EM-seq libraries. In ClinVar gene regions, where both methods provide more similar overall coverage, the percent of miscalls is also similar between EM-seq and to WGBS libraries ( Figure 7).…”

“…Previously published [38] libraries from 10, 50, and 200 ng of genomic DNA from the NA12878 cell line (Coriel) were analyzed. This input DNA was supplemented with a small amount of lambda phage DNA (NEB #N3011), and a pUC19 plasmid (NEB #N3041) treated with M.SssI CpG Methyltransferase (NEB #M0226).…”

“…It is also possible to use an enzymatic method, EM-seq, employing TET2 to oxidize 5-methylcytosine and an APOBEC enzyme to deaminate unmodied cytosines to uracil. While sodium bisulte treatment produces Treat with TET2 -> APOBEC Sequencing Methods Analysis Methods double strand breaks and other DNA damage, this enzymatic method deaminates with more precision [38] and longer, better mappable fragments.…”