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Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.

Luo GZ, Wang F, Weng X, Chen K, Hao Z, Yu M, Deng X, Liu J, He C - Nat Commun (2016)

Bottom Line: Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark.DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches.Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.

ABSTRACT
Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N(6)-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

No MeSH data available.


Related in: MedlinePlus

Detection of 6mA by DA-6mA-seq.(a) Flowchart of DA-6mA-seq. DpnI cleaves fully methylated G(6mA)TC sites, whereas the cleavage of DpnII is hindered by hemi- or fully-methylated 6mA. After treatment with restriction enzyme, DNA segments are further sheared by sonication to ∼300 bp followed by standard Illumina DNA library construction procedures. (b) DA-6mA-seq identifies consistent 6mA sites as reported previously8. (c) The genomic distribution of 6mA sites in promoter, genic and intergenic regions. Promoter is defined as −1,000 to +1,000 bp region around transcription start sites (TSS). (d) The periodic distribution pattern of base-resolution 6mA sites identified by DA-6mA-seq around TSS.
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f1: Detection of 6mA by DA-6mA-seq.(a) Flowchart of DA-6mA-seq. DpnI cleaves fully methylated G(6mA)TC sites, whereas the cleavage of DpnII is hindered by hemi- or fully-methylated 6mA. After treatment with restriction enzyme, DNA segments are further sheared by sonication to ∼300 bp followed by standard Illumina DNA library construction procedures. (b) DA-6mA-seq identifies consistent 6mA sites as reported previously8. (c) The genomic distribution of 6mA sites in promoter, genic and intergenic regions. Promoter is defined as −1,000 to +1,000 bp region around transcription start sites (TSS). (d) The periodic distribution pattern of base-resolution 6mA sites identified by DA-6mA-seq around TSS.

Mentions: Because DpnI is known to cut methylated G(6mA)TC, we envisioned a restriction enzyme-assisted, high-throughput sequencing approach to identify single 6mA sites genome-wide (Fig. 1a) and validated it using Chlamydomonas. Genomic DNA from log-phase sample cultured under constant light was extracted and purified. After treatment with DpnI, long DNA fragments were sonicated to ∼300 bp in length, and then a standard Illumina DNA library was constructed for NGS sequencing. After testing different amounts of input DNA, we were able to use as low as 10 ng input DNA for the digestion step and subsequent library construction (Supplementary Fig. 1). The G(6mA)TC sites should be cleaved by DpnI, and represented as the reads ends in the sequencing output. We developed a bioinformatics pipeline to interrogate the methylation status of each A at GATC context. To overcome the false-positive sites generated by random shearing, a binomial probability distribution was used to determine the cutoff of minimum read ends at GATC sites where the A site could be identified as 6mA while maintaining a false-positive rate below 1%; therefore, <1% of identified 6mA sites might be erroneously assigned. A parallel library was also constructed by using DpnII, which specifically cleaves unmethylated GATC sites (Fig. 1a).


Characterization of eukaryotic DNA N(6)-methyladenine by a highly sensitive restriction enzyme-assisted sequencing.

Luo GZ, Wang F, Weng X, Chen K, Hao Z, Yu M, Deng X, Liu J, He C - Nat Commun (2016)

Detection of 6mA by DA-6mA-seq.(a) Flowchart of DA-6mA-seq. DpnI cleaves fully methylated G(6mA)TC sites, whereas the cleavage of DpnII is hindered by hemi- or fully-methylated 6mA. After treatment with restriction enzyme, DNA segments are further sheared by sonication to ∼300 bp followed by standard Illumina DNA library construction procedures. (b) DA-6mA-seq identifies consistent 6mA sites as reported previously8. (c) The genomic distribution of 6mA sites in promoter, genic and intergenic regions. Promoter is defined as −1,000 to +1,000 bp region around transcription start sites (TSS). (d) The periodic distribution pattern of base-resolution 6mA sites identified by DA-6mA-seq around TSS.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835550&req=5

f1: Detection of 6mA by DA-6mA-seq.(a) Flowchart of DA-6mA-seq. DpnI cleaves fully methylated G(6mA)TC sites, whereas the cleavage of DpnII is hindered by hemi- or fully-methylated 6mA. After treatment with restriction enzyme, DNA segments are further sheared by sonication to ∼300 bp followed by standard Illumina DNA library construction procedures. (b) DA-6mA-seq identifies consistent 6mA sites as reported previously8. (c) The genomic distribution of 6mA sites in promoter, genic and intergenic regions. Promoter is defined as −1,000 to +1,000 bp region around transcription start sites (TSS). (d) The periodic distribution pattern of base-resolution 6mA sites identified by DA-6mA-seq around TSS.
Mentions: Because DpnI is known to cut methylated G(6mA)TC, we envisioned a restriction enzyme-assisted, high-throughput sequencing approach to identify single 6mA sites genome-wide (Fig. 1a) and validated it using Chlamydomonas. Genomic DNA from log-phase sample cultured under constant light was extracted and purified. After treatment with DpnI, long DNA fragments were sonicated to ∼300 bp in length, and then a standard Illumina DNA library was constructed for NGS sequencing. After testing different amounts of input DNA, we were able to use as low as 10 ng input DNA for the digestion step and subsequent library construction (Supplementary Fig. 1). The G(6mA)TC sites should be cleaved by DpnI, and represented as the reads ends in the sequencing output. We developed a bioinformatics pipeline to interrogate the methylation status of each A at GATC context. To overcome the false-positive sites generated by random shearing, a binomial probability distribution was used to determine the cutoff of minimum read ends at GATC sites where the A site could be identified as 6mA while maintaining a false-positive rate below 1%; therefore, <1% of identified 6mA sites might be erroneously assigned. A parallel library was also constructed by using DpnII, which specifically cleaves unmethylated GATC sites (Fig. 1a).

Bottom Line: Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark.DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches.Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Institute for Biophysical Dynamics, Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.

ABSTRACT
Although extensively studied in prokaryotes, the prevalence and significance of DNA N(6)-methyladenine (6mA or m(6)dA) in eukaryotes had been underappreciated until recent studies, which have demonstrated that 6mA regulates gene expression as a potential heritable mark. To interrogate 6mA sites at single-base resolution, we report DA-6mA-seq (DpnI-Assisted N(6)-methylAdenine sequencing), an approach that uses DpnI to cleave methylated adenine sites in duplex DNA. We find that DpnI cuts other sequence motifs besides the canonical GATC restriction sites, thereby expanding the utility of this method. DA-6mA-seq achieves higher sensitivity with nanograms of input DNA and lower sequencing depth than conventional approaches. We study 6mA at base resolution in the Chlamydomonas genome and apply the new method to two other eukaryotic organisms, Plasmodium and Penicillium. Combined with conventional approaches, our method further shows that most 6mA sites are fully methylated on both strands of DNA at various sequence contexts.

No MeSH data available.


Related in: MedlinePlus