Limits...
Single molecule-level detection and long read-based phasing of epigenetic variations in bacterial methylomes.

Beaulaurier J, Zhang XS, Zhu S, Sebra R, Rosenbluh C, Deikus G, Shen N, Munera D, Waldor MK, Chess A, Blaser MJ, Schadt EE, Fang G - Nat Commun (2015)

Bottom Line: Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation.Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity.SMALR is a powerful new tool that enables de novo detection of epigenetic heterogeneity and empowers investigation of its functions in bacterial populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Genomic Sciences and Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York 10029, USA.

ABSTRACT
Beyond its role in host defense, bacterial DNA methylation also plays important roles in the regulation of gene expression, virulence and antibiotic resistance. Bacterial cells in a clonal population can generate epigenetic heterogeneity to increase population-level phenotypic plasticity. Single molecule, real-time (SMRT) sequencing enables the detection of N6-methyladenine and N4-methylcytosine, two major types of DNA modifications comprising the bacterial methylome. However, existing SMRT sequencing-based methods for studying bacterial methylomes rely on a population-level consensus that lacks the single-cell resolution required to observe epigenetic heterogeneity. Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation. Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity. SMALR is a powerful new tool that enables de novo detection of epigenetic heterogeneity and empowers investigation of its functions in bacterial populations.

No MeSH data available.


Related in: MedlinePlus

SMSN score distributions reveal epigenetic heterogeneity.(a) Single molecule, single nucleotide (SMSN) score distributions for multiple bacterium-motif pairs (and the genome-wide motif count, N, of each motif) that exhibit near complete methylation, along with a non-methylated motif for comparison. (b) SMSN score distributions for multiple bacterium-motif pairs that display significant non-methylated fractions. The H. pylori J99 motifs show minor variation in the SMSN associated with each peak due to subtle differences in the chemistry version used for SMRT sequencing of the native and WGA samples. (c) SMSN interrogation of 5′-GANTC methylation at five genomic positions (columns) in a synchronized C. crescentus culture during a single round of DNA replication. Five time points (minutes post-synchronization; rows) provide snapshots of the bidirectional progression of the replication forks from the origin of replication (Cori) to the terminus (Ter). Grey wedges in the chromosome schematics show the 200-kb genomic regions where the SMSN scores are queried for each time point. Two regions are on either side of the Cori: (i) Cori - 0.1 Mbp and (ii) Cori+0.1 Mbp. Another two are halfway between Cori and Ter: (iii) Cori - 1 Mbp and (iv) Cori+1 Mbp. The final region covers the terminus: (v) Ter. Light (hemimethylated) to dark (fully methylated) colour shading in the schematic illustrates the approximate position of the replication fork at each time point. The bimodal distributions of approximate SMSN scores (Methods) reveal the progressive hemi-methylation of 5′-GANTC sites following the passage of the replication forks. Hemimethylated sites cannot transition back to full methylation until the MTase gene, ccrM, is transcribed, which does not occur until late in the replication process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490391&req=5

f3: SMSN score distributions reveal epigenetic heterogeneity.(a) Single molecule, single nucleotide (SMSN) score distributions for multiple bacterium-motif pairs (and the genome-wide motif count, N, of each motif) that exhibit near complete methylation, along with a non-methylated motif for comparison. (b) SMSN score distributions for multiple bacterium-motif pairs that display significant non-methylated fractions. The H. pylori J99 motifs show minor variation in the SMSN associated with each peak due to subtle differences in the chemistry version used for SMRT sequencing of the native and WGA samples. (c) SMSN interrogation of 5′-GANTC methylation at five genomic positions (columns) in a synchronized C. crescentus culture during a single round of DNA replication. Five time points (minutes post-synchronization; rows) provide snapshots of the bidirectional progression of the replication forks from the origin of replication (Cori) to the terminus (Ter). Grey wedges in the chromosome schematics show the 200-kb genomic regions where the SMSN scores are queried for each time point. Two regions are on either side of the Cori: (i) Cori - 0.1 Mbp and (ii) Cori+0.1 Mbp. Another two are halfway between Cori and Ter: (iii) Cori - 1 Mbp and (iv) Cori+1 Mbp. The final region covers the terminus: (v) Ter. Light (hemimethylated) to dark (fully methylated) colour shading in the schematic illustrates the approximate position of the replication fork at each time point. The bimodal distributions of approximate SMSN scores (Methods) reveal the progressive hemi-methylation of 5′-GANTC sites following the passage of the replication forks. Hemimethylated sites cannot transition back to full methylation until the MTase gene, ccrM, is transcribed, which does not occur until late in the replication process.

Mentions: We applied the SMSN analysis to six bacterial methylomes that were recently sequenced353738 or were specifically sequenced for this study (Supplementary Table 1). We first detected methylation motifs based on existing methods243537 and divided them into two groups based on the global distribution of SMSN scores. In the first group (Fig. 3a), most (>95%) motif sites were methylated, with only a small proportion non-methylated, likely due to competitive binding between the MTases and other DNA-binding proteins such as transcription factors23111314. In the second group (Fig. 3b), a substantial (>5%) percentage of motif sites were non-methylated, suggesting the existence of alternative mechanisms that drive methylome heterogeneity. After analysing the SMSN scores for all bacterium-motif pairs (Supplementary Figs 3-8), we observed that while most motifs belong to the first group and do not show extensive non-methylation, the second group includes the 5′-RGATCY motif of C. salexigens and three motifs from Helicobacter pylori J99. Most intriguingly, the H. pylori motif 5′-GWCAY shows a very high (75.3%) percentage of non-methylated sites, which we subsequently investigated using the SMP analysis (see below).


Single molecule-level detection and long read-based phasing of epigenetic variations in bacterial methylomes.

Beaulaurier J, Zhang XS, Zhu S, Sebra R, Rosenbluh C, Deikus G, Shen N, Munera D, Waldor MK, Chess A, Blaser MJ, Schadt EE, Fang G - Nat Commun (2015)

SMSN score distributions reveal epigenetic heterogeneity.(a) Single molecule, single nucleotide (SMSN) score distributions for multiple bacterium-motif pairs (and the genome-wide motif count, N, of each motif) that exhibit near complete methylation, along with a non-methylated motif for comparison. (b) SMSN score distributions for multiple bacterium-motif pairs that display significant non-methylated fractions. The H. pylori J99 motifs show minor variation in the SMSN associated with each peak due to subtle differences in the chemistry version used for SMRT sequencing of the native and WGA samples. (c) SMSN interrogation of 5′-GANTC methylation at five genomic positions (columns) in a synchronized C. crescentus culture during a single round of DNA replication. Five time points (minutes post-synchronization; rows) provide snapshots of the bidirectional progression of the replication forks from the origin of replication (Cori) to the terminus (Ter). Grey wedges in the chromosome schematics show the 200-kb genomic regions where the SMSN scores are queried for each time point. Two regions are on either side of the Cori: (i) Cori - 0.1 Mbp and (ii) Cori+0.1 Mbp. Another two are halfway between Cori and Ter: (iii) Cori - 1 Mbp and (iv) Cori+1 Mbp. The final region covers the terminus: (v) Ter. Light (hemimethylated) to dark (fully methylated) colour shading in the schematic illustrates the approximate position of the replication fork at each time point. The bimodal distributions of approximate SMSN scores (Methods) reveal the progressive hemi-methylation of 5′-GANTC sites following the passage of the replication forks. Hemimethylated sites cannot transition back to full methylation until the MTase gene, ccrM, is transcribed, which does not occur until late in the replication process.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490391&req=5

f3: SMSN score distributions reveal epigenetic heterogeneity.(a) Single molecule, single nucleotide (SMSN) score distributions for multiple bacterium-motif pairs (and the genome-wide motif count, N, of each motif) that exhibit near complete methylation, along with a non-methylated motif for comparison. (b) SMSN score distributions for multiple bacterium-motif pairs that display significant non-methylated fractions. The H. pylori J99 motifs show minor variation in the SMSN associated with each peak due to subtle differences in the chemistry version used for SMRT sequencing of the native and WGA samples. (c) SMSN interrogation of 5′-GANTC methylation at five genomic positions (columns) in a synchronized C. crescentus culture during a single round of DNA replication. Five time points (minutes post-synchronization; rows) provide snapshots of the bidirectional progression of the replication forks from the origin of replication (Cori) to the terminus (Ter). Grey wedges in the chromosome schematics show the 200-kb genomic regions where the SMSN scores are queried for each time point. Two regions are on either side of the Cori: (i) Cori - 0.1 Mbp and (ii) Cori+0.1 Mbp. Another two are halfway between Cori and Ter: (iii) Cori - 1 Mbp and (iv) Cori+1 Mbp. The final region covers the terminus: (v) Ter. Light (hemimethylated) to dark (fully methylated) colour shading in the schematic illustrates the approximate position of the replication fork at each time point. The bimodal distributions of approximate SMSN scores (Methods) reveal the progressive hemi-methylation of 5′-GANTC sites following the passage of the replication forks. Hemimethylated sites cannot transition back to full methylation until the MTase gene, ccrM, is transcribed, which does not occur until late in the replication process.
Mentions: We applied the SMSN analysis to six bacterial methylomes that were recently sequenced353738 or were specifically sequenced for this study (Supplementary Table 1). We first detected methylation motifs based on existing methods243537 and divided them into two groups based on the global distribution of SMSN scores. In the first group (Fig. 3a), most (>95%) motif sites were methylated, with only a small proportion non-methylated, likely due to competitive binding between the MTases and other DNA-binding proteins such as transcription factors23111314. In the second group (Fig. 3b), a substantial (>5%) percentage of motif sites were non-methylated, suggesting the existence of alternative mechanisms that drive methylome heterogeneity. After analysing the SMSN scores for all bacterium-motif pairs (Supplementary Figs 3-8), we observed that while most motifs belong to the first group and do not show extensive non-methylation, the second group includes the 5′-RGATCY motif of C. salexigens and three motifs from Helicobacter pylori J99. Most intriguingly, the H. pylori motif 5′-GWCAY shows a very high (75.3%) percentage of non-methylated sites, which we subsequently investigated using the SMP analysis (see below).

Bottom Line: Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation.Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity.SMALR is a powerful new tool that enables de novo detection of epigenetic heterogeneity and empowers investigation of its functions in bacterial populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Genomic Sciences and Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York 10029, USA.

ABSTRACT
Beyond its role in host defense, bacterial DNA methylation also plays important roles in the regulation of gene expression, virulence and antibiotic resistance. Bacterial cells in a clonal population can generate epigenetic heterogeneity to increase population-level phenotypic plasticity. Single molecule, real-time (SMRT) sequencing enables the detection of N6-methyladenine and N4-methylcytosine, two major types of DNA modifications comprising the bacterial methylome. However, existing SMRT sequencing-based methods for studying bacterial methylomes rely on a population-level consensus that lacks the single-cell resolution required to observe epigenetic heterogeneity. Here, we present SMALR (single-molecule modification analysis of long reads), a novel framework for single molecule-level detection and phasing of DNA methylation. Using seven bacterial strains, we show that SMALR yields significantly improved resolution and reveals distinct types of epigenetic heterogeneity. SMALR is a powerful new tool that enables de novo detection of epigenetic heterogeneity and empowers investigation of its functions in bacterial populations.

No MeSH data available.


Related in: MedlinePlus