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Editor | xi The most extensive DNA methylation in the human genome is 5-methylcytosine (5mC), and there are three common DNA methylations in bacteria: N6-methyladenie (6mA), N4-methylcytosine (4mC) and 5mC.
In 2012, Ganghe Fang's team at Mount Sinai School of Medicine in New York used third-generation single-molecule real-time sequencing technology (Pacific Biosciences, PacBio) to map the 6mA epigenome of bacteria for the first time, and found that 6mA can directly or indirectly mediate more than 1,000 genes in E.
coli The transcription level [1].
In the past nine years, the epigenetic group of >4000 strains of bacteria has been deciphered: 95% of bacteria have DNA methylation; on average, each bacteria has three different active DNA methylases and corresponding methylation.
Recognition sequence (methylation motifs), there are more than 20 at most, such as Helicobacter pylori [2].
In 2018, Fang Gang's group at Icahn Medical College of Mount Sinai published a review in Nature Reviews Genetics [3] A detailed summary of various sequencing technologies, especially the third-generation sequencing technology in the study of bacterial epigenetics Application, as well as the multiple important functions of epigenetics in bacterial gene expression and pathogenic bacteria (see BioArt report: Nat Rev Genet published Fang Gang group bacterial epigenetic review paper).
On April 5, 2021, Fang Gang’s group published an article Discovering multiple types of DNA methylation from bacteria and microbiome using nanopore sequencing in Nature Methods, and developed a new method NanoDisco, which uses nanopore sequencing to simultaneously detect multiple types of bacterial DNA methylation motifs , And use the diversity of bacterial DNA methylation to enhance the clarity of flora analysis.
This research has three key points: 1.
Deep understanding of DNA methylation detection by nanopore sequencing: The epigenetic group of >4000 strains of deciphered bacteria so far is almost entirely based on the PacBio third-generation sequencing platform.
This platform has very high reliability and sensitivity for 6mA and 4mC motifs in bacteria, but the detection efficiency for 5mC is not good.
Another third-generation sequencing technology (Nanopore nanopore sequencing) has gradually been widely used in the detection of 5mC in the human genome, but most of the existing technologies are specifically aimed at the detection of 5mC in CpG.
Fang Gang’s research team analyzed a large number of bacterial 5mC motifs and found that the 5mC of different sequence motifs has high signal heterogeneity in nanopore sequencing (Figure 1); similarly, 6mA and 4mC signals also have high differences.
Qualitative.
In other words, the existing nanopore sequencing detection methods for CpG 5mC (or a small number of specific sequences) cannot be effectively applied to a wider range of DNA methylation detection, that is, it cannot effectively detect the three common DNA methyl groups in bacteria.
.
Figure 1: The signals of 6mA, 4mC and 5mC in different sequence motifs have high heterogeneity in nanopore sequencing.
2.
De novo (no prior knowledge required) discovered three new methods of DNA methylation in bacteria.
The first step in bacterial DNA methylation analysis is de novo to find all methylation motifs.
What everyone is more familiar with is G6mATC and C5mCWGG which are widely present in E.
coli.
On average, each bacterial strain has three different methylation motifs, but there are more than 20, such as Helicobacter pylori.
For an unknown motif, it is necessary to identify the specific methylation type (6mA, 4mC, or 5mC) and find the methylated base (Figure 2).
Taking into account some of the characteristics of bacterial DNA methylation, the design of the new method NanoDisco uses a multi-label classification machine learning architecture.
After extensive verification, NanoDisco can reliably de novo find 6mA, 4mC, 5mC motifs and methylation sites in bacteria and flora.
The author believes that this function will greatly help a broader and more complete study of bacterial DNA methylation.
Indeed, from actual bacteria and flora samples, the article also found that many bacteria actually have multiple 5mC motifs, far exceeding the estimates of previous studies, thus creating many new research opportunities.
Figure 2: The new method NanoDisco also solves the two fundamental problems encountered in the discovery of DNA methylation motifs using nanopore sequencing de novo: 1) Identify the methylation typing; 2) Find the specific motif in the motif 2.
Methylated base (fine mapping) 3.
Use bacterial DNA methylation diversity to enhance flora analysis.
Fang Gang’s team cleverly used bacterial DNA methylation diversity as a natural epigenetic barcode in 2017 to distinguish bacterial species and strains with highly similar genomes, so as to analyze the flora with high definition [4] (see BioArt for details) Text: Chinese scholars open the functional exploration of DNA methylation in the study of microbial communitiesNat Biotech).
The research at that time was based on the PacBio sequencing platform, so it could only effectively use 6mA and 4mC methylation in bacteria.
The new method developed in this new research effectively handles some essential differences between PacBio sequencing and nanopore sequencing, so as to realize the use of three types of methylation (6mA, 4mC, 5mC) to better distinguish different bacteria in the flora ( Figure 3), associate mobile genetic elements (Mobile Genetic Elements, such as plasmids) with their hosts, and detect errors in metagenomic assembly.
Figure 3: Simultaneous use of three bacterial DNA methylation diversity as a natural epigenetic barcode for high-resolution metagenomic analysis.
The article also compares two common third-generation sequencing technologies: PacBio's third-generation sequencing technology is not good at 5mC is detected in diverse sequence motifs, but for the detection of bacterial 6mA and 4mC motifs, PacBio sequencing is still superior to nanopore sequencing, that is, the two platforms are still complementary; researchers should choose a sequencing platform based on specific issues.
The article also emphasizes that NanoDisco focuses on the analysis of bacterial methylation at the motif level rather than the analysis of individual methylation sites, because for the latter, the accuracy of nanopore sequencing varies greatly between different motifs.
In recent years, there have been many interesting findings in the study of bacterial methylation: many pathogenic bacteria use epigenetic mechanisms to regulate genes and phenotypes related to infectious diseases (toxicity, biofilm, sporulation, etc.
) [3 , 5].
However, bacterial epigenetics and epigenetics are still in its infancy; there are many fundamental problems and clinical applications that need to be resolved.
It is hoped that this new method can promote the study of bacterial DNA methylation and help more reliable flora analysis.
Software download: https://github.
com/fanglab/nanodisco Instructions for use: https://nanodisco.
readthedocs.
io/en/latest/overview.
html FAQ: https://nanodisco.
readthedocs.
io/en/ latest/faq.
html (It is worth mentioning that: the author emphasizes that this method focuses on the analysis of bacteria and flora, and does not recommend its use in eukaryotes) At the same time, the Fang Gang research group of Icahn School of Medicine at Mount Sinai is working Recruiting postdoctoral fellows to conduct bacterial epigenetics and the application of human flora in disease research. Research group webpage: http://fanglab.
bio/ resume delivery (if interested, please send your resume and other materials to): https://jinshuju.
net/f/ZqXwZt or scan the QR code to deliver the original resume link: https://doi.
org/10.
1038/s41592-021-01109-3 Platemaker: Eleven References [1] Fang G, Munera D, Friedman DI, Mandlik A, Chao MC, Banerjee O, Feng Z, Losic B , Mahajan MC, Jabado OJ.
2012.
Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.
Nat Biotechnol 30: 1232–1239.
[2] Sánchez-Romero, María A.
, and Josep Casadesús.
"The bacterial epigenome.
" Nature Reviews Microbiology 18.
1 (2020): 7-20.
[3] Beaulaurier J, Schadt EE, Fang G, Nature Reviews Genetics, 20, pages157–172(2019)[4] Beaulaurier J , Zhu S, Deikus G, Mogno I, Zhang XS, Davis-Richardson A, Canepa R, Triplett EW, Faith JJ, Sebra R, Schadt EE & Fang G,Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation, Nature Biotechnology, 10.
1038/nbt.
4037, 2018[5] Oliveira PH, Kim A, Sekulovic O, Garrett EM, Trzilova D, Mead EA, Pak T, Zhu S , Deikus S, .
.
.
, Patel G, Wallach F, Hamula C, Huprikar S, Roberts RJ, Schadt EE, Sebra R, van Bakel H, Kasarskis A, Tamayo R, Shen A#, Fang G#, Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis, Nature Microbiology, 5, pages 166–180, 2020 Reprint instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author owns all Legal rights, offenders must be investigated.
Kasarskis A, Tamayo R, Shen A#, Fang G#, Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis, Nature Microbiology, 5, pages 166–180, 2020 Reprint Notice [Non-original article] The copyright of this article belongs to the article Owned by the author, personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
Kasarskis A, Tamayo R, Shen A#, Fang G#, Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis, Nature Microbiology, 5, pages 166–180, 2020 Reprint Notice [Non-original article] The copyright of this article belongs to the article Owned by the author, personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
In 2012, Ganghe Fang's team at Mount Sinai School of Medicine in New York used third-generation single-molecule real-time sequencing technology (Pacific Biosciences, PacBio) to map the 6mA epigenome of bacteria for the first time, and found that 6mA can directly or indirectly mediate more than 1,000 genes in E.
coli The transcription level [1].
In the past nine years, the epigenetic group of >4000 strains of bacteria has been deciphered: 95% of bacteria have DNA methylation; on average, each bacteria has three different active DNA methylases and corresponding methylation.
Recognition sequence (methylation motifs), there are more than 20 at most, such as Helicobacter pylori [2].
In 2018, Fang Gang's group at Icahn Medical College of Mount Sinai published a review in Nature Reviews Genetics [3] A detailed summary of various sequencing technologies, especially the third-generation sequencing technology in the study of bacterial epigenetics Application, as well as the multiple important functions of epigenetics in bacterial gene expression and pathogenic bacteria (see BioArt report: Nat Rev Genet published Fang Gang group bacterial epigenetic review paper).
On April 5, 2021, Fang Gang’s group published an article Discovering multiple types of DNA methylation from bacteria and microbiome using nanopore sequencing in Nature Methods, and developed a new method NanoDisco, which uses nanopore sequencing to simultaneously detect multiple types of bacterial DNA methylation motifs , And use the diversity of bacterial DNA methylation to enhance the clarity of flora analysis.
This research has three key points: 1.
Deep understanding of DNA methylation detection by nanopore sequencing: The epigenetic group of >4000 strains of deciphered bacteria so far is almost entirely based on the PacBio third-generation sequencing platform.
This platform has very high reliability and sensitivity for 6mA and 4mC motifs in bacteria, but the detection efficiency for 5mC is not good.
Another third-generation sequencing technology (Nanopore nanopore sequencing) has gradually been widely used in the detection of 5mC in the human genome, but most of the existing technologies are specifically aimed at the detection of 5mC in CpG.
Fang Gang’s research team analyzed a large number of bacterial 5mC motifs and found that the 5mC of different sequence motifs has high signal heterogeneity in nanopore sequencing (Figure 1); similarly, 6mA and 4mC signals also have high differences.
Qualitative.
In other words, the existing nanopore sequencing detection methods for CpG 5mC (or a small number of specific sequences) cannot be effectively applied to a wider range of DNA methylation detection, that is, it cannot effectively detect the three common DNA methyl groups in bacteria.
.
Figure 1: The signals of 6mA, 4mC and 5mC in different sequence motifs have high heterogeneity in nanopore sequencing.
2.
De novo (no prior knowledge required) discovered three new methods of DNA methylation in bacteria.
The first step in bacterial DNA methylation analysis is de novo to find all methylation motifs.
What everyone is more familiar with is G6mATC and C5mCWGG which are widely present in E.
coli.
On average, each bacterial strain has three different methylation motifs, but there are more than 20, such as Helicobacter pylori.
For an unknown motif, it is necessary to identify the specific methylation type (6mA, 4mC, or 5mC) and find the methylated base (Figure 2).
Taking into account some of the characteristics of bacterial DNA methylation, the design of the new method NanoDisco uses a multi-label classification machine learning architecture.
After extensive verification, NanoDisco can reliably de novo find 6mA, 4mC, 5mC motifs and methylation sites in bacteria and flora.
The author believes that this function will greatly help a broader and more complete study of bacterial DNA methylation.
Indeed, from actual bacteria and flora samples, the article also found that many bacteria actually have multiple 5mC motifs, far exceeding the estimates of previous studies, thus creating many new research opportunities.
Figure 2: The new method NanoDisco also solves the two fundamental problems encountered in the discovery of DNA methylation motifs using nanopore sequencing de novo: 1) Identify the methylation typing; 2) Find the specific motif in the motif 2.
Methylated base (fine mapping) 3.
Use bacterial DNA methylation diversity to enhance flora analysis.
Fang Gang’s team cleverly used bacterial DNA methylation diversity as a natural epigenetic barcode in 2017 to distinguish bacterial species and strains with highly similar genomes, so as to analyze the flora with high definition [4] (see BioArt for details) Text: Chinese scholars open the functional exploration of DNA methylation in the study of microbial communitiesNat Biotech).
The research at that time was based on the PacBio sequencing platform, so it could only effectively use 6mA and 4mC methylation in bacteria.
The new method developed in this new research effectively handles some essential differences between PacBio sequencing and nanopore sequencing, so as to realize the use of three types of methylation (6mA, 4mC, 5mC) to better distinguish different bacteria in the flora ( Figure 3), associate mobile genetic elements (Mobile Genetic Elements, such as plasmids) with their hosts, and detect errors in metagenomic assembly.
Figure 3: Simultaneous use of three bacterial DNA methylation diversity as a natural epigenetic barcode for high-resolution metagenomic analysis.
The article also compares two common third-generation sequencing technologies: PacBio's third-generation sequencing technology is not good at 5mC is detected in diverse sequence motifs, but for the detection of bacterial 6mA and 4mC motifs, PacBio sequencing is still superior to nanopore sequencing, that is, the two platforms are still complementary; researchers should choose a sequencing platform based on specific issues.
The article also emphasizes that NanoDisco focuses on the analysis of bacterial methylation at the motif level rather than the analysis of individual methylation sites, because for the latter, the accuracy of nanopore sequencing varies greatly between different motifs.
In recent years, there have been many interesting findings in the study of bacterial methylation: many pathogenic bacteria use epigenetic mechanisms to regulate genes and phenotypes related to infectious diseases (toxicity, biofilm, sporulation, etc.
) [3 , 5].
However, bacterial epigenetics and epigenetics are still in its infancy; there are many fundamental problems and clinical applications that need to be resolved.
It is hoped that this new method can promote the study of bacterial DNA methylation and help more reliable flora analysis.
Software download: https://github.
com/fanglab/nanodisco Instructions for use: https://nanodisco.
readthedocs.
io/en/latest/overview.
html FAQ: https://nanodisco.
readthedocs.
io/en/ latest/faq.
html (It is worth mentioning that: the author emphasizes that this method focuses on the analysis of bacteria and flora, and does not recommend its use in eukaryotes) At the same time, the Fang Gang research group of Icahn School of Medicine at Mount Sinai is working Recruiting postdoctoral fellows to conduct bacterial epigenetics and the application of human flora in disease research. Research group webpage: http://fanglab.
bio/ resume delivery (if interested, please send your resume and other materials to): https://jinshuju.
net/f/ZqXwZt or scan the QR code to deliver the original resume link: https://doi.
org/10.
1038/s41592-021-01109-3 Platemaker: Eleven References [1] Fang G, Munera D, Friedman DI, Mandlik A, Chao MC, Banerjee O, Feng Z, Losic B , Mahajan MC, Jabado OJ.
2012.
Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.
Nat Biotechnol 30: 1232–1239.
[2] Sánchez-Romero, María A.
, and Josep Casadesús.
"The bacterial epigenome.
" Nature Reviews Microbiology 18.
1 (2020): 7-20.
[3] Beaulaurier J, Schadt EE, Fang G, Nature Reviews Genetics, 20, pages157–172(2019)[4] Beaulaurier J , Zhu S, Deikus G, Mogno I, Zhang XS, Davis-Richardson A, Canepa R, Triplett EW, Faith JJ, Sebra R, Schadt EE & Fang G,Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation, Nature Biotechnology, 10.
1038/nbt.
4037, 2018[5] Oliveira PH, Kim A, Sekulovic O, Garrett EM, Trzilova D, Mead EA, Pak T, Zhu S , Deikus S, .
.
.
, Patel G, Wallach F, Hamula C, Huprikar S, Roberts RJ, Schadt EE, Sebra R, van Bakel H, Kasarskis A, Tamayo R, Shen A#, Fang G#, Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis, Nature Microbiology, 5, pages 166–180, 2020 Reprint instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author owns all Legal rights, offenders must be investigated.
Kasarskis A, Tamayo R, Shen A#, Fang G#, Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis, Nature Microbiology, 5, pages 166–180, 2020 Reprint Notice [Non-original article] The copyright of this article belongs to the article Owned by the author, personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
Kasarskis A, Tamayo R, Shen A#, Fang G#, Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis, Nature Microbiology, 5, pages 166–180, 2020 Reprint Notice [Non-original article] The copyright of this article belongs to the article Owned by the author, personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
