Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA
Abstract Background Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases o...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-06-01
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| Series: | BMC Genomics |
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| Online Access: | http://link.springer.com/article/10.1186/s12864-019-5754-6 |
| _version_ | 1818083764932182016 |
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| author | Zach K. O’Brown Konstantinos Boulias Jie Wang Simon Yuan Wang Natasha M. O’Brown Ziyang Hao Hiroki Shibuya Paul-Enguerrand Fady Yang Shi Chuan He Sean G. Megason Tao Liu Eric L. Greer |
| author_facet | Zach K. O’Brown Konstantinos Boulias Jie Wang Simon Yuan Wang Natasha M. O’Brown Ziyang Hao Hiroki Shibuya Paul-Enguerrand Fady Yang Shi Chuan He Sean G. Megason Tao Liu Eric L. Greer |
| author_sort | Zach K. O’Brown |
| collection | DOAJ |
| description | Abstract Background Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background. Conclusions Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques. |
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| format | Article |
| id | doaj.art-5f5c2596b8554fddb265cdf456fcd9f1 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-10T19:43:11Z |
| publishDate | 2019-06-01 |
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| spelling | doaj.art-5f5c2596b8554fddb265cdf456fcd9f12022-12-22T01:35:58ZengBMCBMC Genomics1471-21642019-06-0120111510.1186/s12864-019-5754-6Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNAZach K. O’Brown0Konstantinos Boulias1Jie Wang2Simon Yuan Wang3Natasha M. O’Brown4Ziyang Hao5Hiroki Shibuya6Paul-Enguerrand Fady7Yang Shi8Chuan He9Sean G. Megason10Tao Liu11Eric L. Greer12Division of Newborn Medicine, Boston Children’s HospitalDivision of Newborn Medicine, Boston Children’s HospitalDepartment of Biochemistry and Biostatistics, University at Buffalo Center of Excellence in Bioinformatics & Life SciencesDivision of Newborn Medicine, Boston Children’s HospitalDepartment of Neurobiology, Harvard Medical SchoolDepartment of Chemistry and Howard Hughes Medical Institute, The University of ChicagoDivision of Newborn Medicine, Boston Children’s HospitalDivision of Newborn Medicine, Boston Children’s HospitalDivision of Newborn Medicine, Boston Children’s HospitalDepartment of Chemistry and Howard Hughes Medical Institute, The University of ChicagoDepartment of Systems Biology, Harvard Medical SchoolDepartment of Biochemistry and Biostatistics, University at Buffalo Center of Excellence in Bioinformatics & Life SciencesDivision of Newborn Medicine, Boston Children’s HospitalAbstract Background Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background. Conclusions Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.http://link.springer.com/article/10.1186/s12864-019-5754-6DNA epigenomeDNA N6-methyladenosine6 mADNA N4-methylcytosine4mC |
| spellingShingle | Zach K. O’Brown Konstantinos Boulias Jie Wang Simon Yuan Wang Natasha M. O’Brown Ziyang Hao Hiroki Shibuya Paul-Enguerrand Fady Yang Shi Chuan He Sean G. Megason Tao Liu Eric L. Greer Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA BMC Genomics DNA epigenome DNA N6-methyladenosine 6 mA DNA N4-methylcytosine 4mC |
| title | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA |
| title_full | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA |
| title_fullStr | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA |
| title_full_unstemmed | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA |
| title_short | Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA |
| title_sort | sources of artifact in measurements of 6ma and 4mc abundance in eukaryotic genomic dna |
| topic | DNA epigenome DNA N6-methyladenosine 6 mA DNA N4-methylcytosine 4mC |
| url | http://link.springer.com/article/10.1186/s12864-019-5754-6 |
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