Single-Nucleotide Resolution Mapping of N6-Methyladenine in Genomic DNA

N6-Methyladenine (6mA) is a naturally occurring DNA modification in both prokaryotes and eukaryotes. Herein, we developed a deaminase-mediated sequencing (DM-seq) method for genome-wide mapping of 6mA at single-nucleotide resolution. The method capitalizes on the selective deamination of adenine, but not 6mA, in DNA mediated by an evolved adenine deaminase, ABE8e. By employing this method, we achieved genome-wide mapping of 6mA in Escherichia coli and in mammalian mitochondrial DNA (mtDNA) at single-nucleotide resolution. We found that the 6mA sites are mainly located in the GATC motif in the E. coli genome. We also identified 17 6mA sites in mtDNA of HepG2 cells, where all of the 6mA sites are distributed in the heavy strand of mtDNA. We envision that DM-seq will be a valuable tool for uncovering new functions of 6mA in DNA and for exploring its potential roles in mitochondria-related human diseases.

Nitrite treatment and sequencing.S3 Table S1.Sequences of oligonucleotides.S4 Table S2.The mass spectrometry parameters for the analysis of nucleosides.S5 Table S3.Evaluation of the deamination rate of dA in different sequence context of the 314-bp 6mA-DNA by colony sequencing.S6 Table S4.Primers for PCR.

S7-S8
Table S5.Sequencing data of the spike-in DNA in different samples by DM-seq.S9 Table S6.Sequencing data by DM-seq.S10 Table S7.6mA sites commonly identified in HepG2 mtDNA from three replicates by DM-seq method.S11 Nitrite treatment and sequencing Nitrite treatment was carried out according to previously published procedures study. 1 Briefly, 1.0 g of synthetic 314-bp A-DNA (Table S1) in 8 µL H2O was first denatured by heating at 95 o C for 10 min, followed by chilling on ice for 5 min.The denatured DNA was then incubated with 15 µL of fresh 2.0 M sodium nitrite and 7 µL 10% acetic acid (v/v) at 37 o C for 5 h.The resulting DNA was purified by Oligo Clean & Concentrator kit (Zymo Research) and subjected to Sanger sequencing and colony sequencing.Note: p, 5′-phosphorylation.S1) was treated by nitrite according to previously described procedures followed by sequencing.

Figure S1 .S23Figure S13 .Figure S14 .Figure S15 .Figure S16 .Figure S17 .Figure S18 .
Figure S1.SDS-PAGE analysis of the purified ABE8e protein.S12 Figure S2.Quantitative evaluation of the Endo V cleavage assay using the mixture of 60-mer single I-DNA and 60-mer single A-DNA.S13 Figure S3.Quantitative evaluation of the deamination of adenine in DNA by ABE8e using the mixture of 60-mer single A-DNA and 60-mer single 6mA-DNA.S14 Figure S4.Evaluation of the deamination of adenine in the 60-mer single A-DNA by ABE8e at different times (0, 1, 5, 10, and 30 min).S15 Figure S5.Evaluation of the normal nucleosides in DNA upon ABE8e treatment by LC-ESI-MS/MS.S16 Figure S6.Evaluation of the resistance of 6mA to ABE8e.S17 Figure S7.Preparation of 314-bp 6mA-DNA using dam methyltransferase.S18 Figure S8.Colony sequencing using 314-bp 6mA-DNA after ABE8e treatment.S19 Figure S9.Evaluation of the quantitative capability of DM-seq using synthesized DNA.S20 Figure S10.Evaluation of the identification of 6mA sites in complex sequence context.S21 Figure S11.Evaluation of the nitrite sequencing.S22 Figure S12.Evaluation of the levels of dA, dI and 6mA in DNA from wild-type E. coli strain K12 (Dam+) or dam deficient E. coli strain SCS110 (Dam-) after ABE8e treatment by LC-ESI-MS/MS analysis.

Figure S2 .
Figure S2.Quantitative evaluation of the Endo V cleavage assay using the mixture of 60-mer single I-DNA and 60-mer single A-DNA.(A) Single I-DNA and single A-DNA were mixed at different molar ratios (0, 20, 40, 60, 80, and 100% of single I-DNA) and then treated with Endo V at 37°C for 1 h.The resulting DNA was analyzed by polyacrylamide gel electrophoresis.(B) The cleaved fractions of single I-DNA were plotted against the percentage of single I-DNA.The slope of 0.99 indicated that the levels of single I-DNA in DNA mixture could be quantitatively obtained using the Endo V cleavage assay.

Figure S3 .
Figure S3.Quantitative evaluation of the deamination of adenine in DNA by ABE8e using the mixture of 60-mer single A-DNA and 60-mer single 6mA-DNA.(A) Analysis of the deamination of adenine by ABE8e with Endo V cleavage assay.Single A-DNA and single 6mA-DNA were mixed at different molar ratios (0, 20, 40, 60, 80, and 100% of single 6mA-DNA) and then treated with ABE8e at 37°C for 30 min.The resulting DNA was incubated with Endo V followed by polyacrylamide gel electrophoresis analysis.(B) The cleaved fractions of DNA were plotted against the percentage of single 6mA-DNA.The slope of -0.99 indicated that the levels of single 6mA-DNA in the DNA mixtures could be quantitatively obtained using the Endo V assay.

Figure S4 .
Figure S4.Evaluation of the deamination of adenine in the 60-mer single A-DNA by ABE8e at different times (0, 1, 5, 10, and 30 min).Endo V cleavage assay was used to evaluate the deamination of adenine.

Figure S5 .
Figure S5.Evaluation of the normal nucleosides in DNA upon ABE8e treatment by LC-ESI-MS/MS.(A) Extracted-ion chromatograms of dC with or without ABE8e treatment in single A-DNA or single 6mA-DNA.(B) Extracted-ion chromatograms of dG with or without ABE8e treatment in single A-DNA or single 6mA-DNA.(C) Extracted-ion chromatograms of dT with or without ABE8e treatment in single A-DNA or single 6mA-DNA.

Figure S6 .
Figure S6.Evaluation of the resistance of 6mA to ABE8e.(A) LC-ESI-MS/MS analysis of the remaining percentage of 6mA upon incubation with different concentrations of ABE8e at 37 o C for 30 min.(B) LC-ESI-MS/MS analysis of the remaining percentage of 6mA upon incubation with of ABE8e (2.8 μM) at 37 o C for different time intervals.The 60-mer single 6mA-DNA was used for the evaluation.

Figure S7 .
Figure S7.Preparation of 314-bp 6mA-DNA using dam methyltransferase.(A) A schematic diagram showing the methylation of 314-bp A-DNA by dam methyltransferase.Dam methyltransferase can methylate adenine at GATC site in the 314-bp A-DNA to generate the 314bp 6mA-DNA.(B) Analysis of the prepared 314-bp 6mA-DNA by Dpn I and Dpn II digestion.Dpn I cleaves only when its recognition site is methylated, i.e., G6mATC site in DNA; but Dpn I cannot cleave the unmethylated GATC site in DNA.On the contrary, Dpn II cleaves only when its recognition site is unmethylated, i.e., GATC site in DNA; but Dpn II cannot cleave the methylated G6mATC site in DNA.

Figure S8 .
Figure S8.Colony sequencing using 314-bp 6mA-DNA after ABE8e treatment.Fifty-three colonies were picked for sequencing.The 6mA sites in all the 53 clones were read as A. A total of 4863 adenine sites out of 4876 (totally 92 adenines/strand × 53 clones = 4876 adenines) in 314-bp 6mA-DNA were read as G.The deamination rate was calculated to be 99.73%(4863/4876 × 100%).Green, read as A. Yellow, read as G.

Figure S9 .
Figure S9.Evaluation of the quantitative capability of DM-seq using synthesized DNA.A 60-mer A-DNA was mixed with different percentages of 60-mer 6mA-DNA in the same sequence context (Table S1) and the readouts of A and G from the DM-Seq were evaluated.(A) The sequences of 60-mer A-DNA and 60-mer 6mA-DNA.(B) Colony sequencing showing the numbers of A reads and G reads from the mixtures with different 6mA percentages.(C) The measured percentages of A reads were plotted with the theoretical 6mA percentages.

Figure S10 .
Figure S10.Evaluation of the identification of 6mA sites in complex sequence context.A 60-mer DNA containing two 6mA sites (dual 6mA-DNA) in the sequence context of AA6mAA6mAAA (TableS1) was synthesized and subjected to DM-seq analysis.(A) The sequence of 60-mer dual 6mA-DNA.(B) A schematic illustration showing the colony sequencing results from 45 clones.(C) The readouts from evaluated A sites and 6mA sites.All the A sites in this region were efficiently deaminated and read as G in sequencing (deamination rate of A being 99.6%).Both 6mA sites in this region resisted deamination and still read as A (deamination rate of 6mA being 0%).

Figure S11 .
Figure S11.Evaluation of the nitrite sequencing.The 314-bp DNA (TableS1) was treated by nitrite according to previously described procedures followed by sequencing.(A) Direct Sanger sequencing results.(B) Representative colony sequencing results.Some A and C are deaminated and read as G and T, respectively.
Figure S11.Evaluation of the nitrite sequencing.The 314-bp DNA (TableS1) was treated by nitrite according to previously described procedures followed by sequencing.(A) Direct Sanger sequencing results.(B) Representative colony sequencing results.Some A and C are deaminated and read as G and T, respectively.

Figure S12 .
Figure S12.Evaluation of the levels of dA, dI and 6mA in DNA from wild-type E. coli strain K12 (Dam+) or dam deficient E. coli strain SCS110 (Dam-) upon ABE8e treatment by LC-ESI-MS/MS analysis.(A) Extracted-ion chromatograms of dA in DNA from Dam+ or Dam-E.coli cells with or without ABE8e treatment.(B) Extracted-ion chromatograms of dI in DNA from Dam+ or Dam-E.coli cells with or without ABE8e treatment.(C) Extracted-ion chromatograms of 6mA in DNA from Dam+ or Dam-E.coli cells with or without ABE8e treatment.

Figure S14 .
Figure S14.The representative view of 6mA sites between the region of 98809 to 98829 in genome of dam deficient E. coli strain SCS110.Red and blue columns represent the number of reads with A and G at given position, respectively.

Figure S15 .
Figure S15.Circos plot showing the identified G6mATC motif from three replicates.Outer three circles with red bars represent the identified G6mATC motif from three replicates.Inner circle with yellow bars showing the theoretical sites of GATC in wild-type E. coli strain K12.The majority of the yellow lines (GATC sites) were red in all the outer three replicates, indicating consistent identification of 6mA.

Figure S16 .
Figure S16.Evaluation of the real-time qPCR amplification efficiencies.(A) Standard curve for amplification of gene in mtDNA.(B) Standard curve for amplification of GAPDH in genomic DNA.The standard curves were constructed with using a series of amounts of DNA templates (0.001, 0.01, 0.1, 1, 10, and 100 ng) for real-time qPCR.The obtained amplification efficiencies of mtDNA and GAPDH were 90.7% and 90.6%, respectively.

Figure S17 .
Figure S17.The proportion of the number of reads mapped to mtDNA over the number of total clean reads.

Figure S18 .
Figure S18.Confirmation of selected 6mA sites in mtDNA by Sanger sequencing.In Sanger sequencing, the 6mA sites identified in mtDNA by DM-seq were read as A, while other A sites were read as G.
Table of Contents:

Table S2 .
The mass spectrometry parameters for the analysis of nucleosides.DP, declustering potential; EP, entrance potential; CE, collision energy; CXP, collision cell exit potential.

Table S3 .
Evaluation of the deamination rates of dA in different sequence contexts of the 314-bp 6mA-DNA by colony sequencing.

Table S5 .
Sequencing data of the spiked-in DNA in different samples by DM-seq.

Table S7 .
6mA sites commonly identified in HepG2 mtDNA from three replicates by DM-seq method.