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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.


Performance of SMSN level detection of DNA methylation.Multiple metrics showing the performance of the proposed single molecule, single nucleotide (SMSN) detection method. (a) Performance of the approach for detecting 6mA modifications in the 5′-CTGCAG motif of E. coli C227 using three thresholds for minimum single-molecule coverage (covSM). (b) Distribution of the aggregate, single nucleotide (AggSN) and SMSN methylation scores for the partially non-methylated 5′-RGATCY motif in C. salexigens. The bimodal distribution of the SMSN scores enables the accurate and objective estimation of this fraction. (c) Accuracy of SMSN-enabled estimations of the methylated fraction (using covSM≥10) for the 5′-CTGCAG motif of E. coli C227 at various levels of genomic-sequencing coverage.
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f2: Performance of SMSN level detection of DNA methylation.Multiple metrics showing the performance of the proposed single molecule, single nucleotide (SMSN) detection method. (a) Performance of the approach for detecting 6mA modifications in the 5′-CTGCAG motif of E. coli C227 using three thresholds for minimum single-molecule coverage (covSM). (b) Distribution of the aggregate, single nucleotide (AggSN) and SMSN methylation scores for the partially non-methylated 5′-RGATCY motif in C. salexigens. The bimodal distribution of the SMSN scores enables the accurate and objective estimation of this fraction. (c) Accuracy of SMSN-enabled estimations of the methylated fraction (using covSM≥10) for the 5′-CTGCAG motif of E. coli C227 at various levels of genomic-sequencing coverage.

Mentions: An accurate mean log IPD for each nucleotide in a single molecule permits confident estimation of the IPD ratio. The accuracy of the mean log IPD increases as the number of subreads for each molecule increases3334, as each subread provides an independent estimate of the IPD. For a given read length, the number of subreads (that is, single-molecule coverage, covSM) is negatively correlated with the insert size of the sequencing library. To quantify the sensitivity and specificity for SMSN detection, we analysed methylated 5′-CTGCAG sites in a native E. coli O104:H4 C227-11 strain35 (referred to as C227), and a matching WGA sample that is free of methylation (Fig. 2a; Methods). As expected, the sensitivity of the method increased with increasing covSM. For the SMSN approach, covSM≥15 enables detection of 6 mA with a sensitivity of 98.5% and a specificity of 99.5%. Although smaller library sizes can provide higher values of covSM, a minimum library size of 150–200 bp is recommended to avoid loss of genomic DNA during library construction and to facilitate removal of adapter-dimer constructs during purification.


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)

Performance of SMSN level detection of DNA methylation.Multiple metrics showing the performance of the proposed single molecule, single nucleotide (SMSN) detection method. (a) Performance of the approach for detecting 6mA modifications in the 5′-CTGCAG motif of E. coli C227 using three thresholds for minimum single-molecule coverage (covSM). (b) Distribution of the aggregate, single nucleotide (AggSN) and SMSN methylation scores for the partially non-methylated 5′-RGATCY motif in C. salexigens. The bimodal distribution of the SMSN scores enables the accurate and objective estimation of this fraction. (c) Accuracy of SMSN-enabled estimations of the methylated fraction (using covSM≥10) for the 5′-CTGCAG motif of E. coli C227 at various levels of genomic-sequencing coverage.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Performance of SMSN level detection of DNA methylation.Multiple metrics showing the performance of the proposed single molecule, single nucleotide (SMSN) detection method. (a) Performance of the approach for detecting 6mA modifications in the 5′-CTGCAG motif of E. coli C227 using three thresholds for minimum single-molecule coverage (covSM). (b) Distribution of the aggregate, single nucleotide (AggSN) and SMSN methylation scores for the partially non-methylated 5′-RGATCY motif in C. salexigens. The bimodal distribution of the SMSN scores enables the accurate and objective estimation of this fraction. (c) Accuracy of SMSN-enabled estimations of the methylated fraction (using covSM≥10) for the 5′-CTGCAG motif of E. coli C227 at various levels of genomic-sequencing coverage.
Mentions: An accurate mean log IPD for each nucleotide in a single molecule permits confident estimation of the IPD ratio. The accuracy of the mean log IPD increases as the number of subreads for each molecule increases3334, as each subread provides an independent estimate of the IPD. For a given read length, the number of subreads (that is, single-molecule coverage, covSM) is negatively correlated with the insert size of the sequencing library. To quantify the sensitivity and specificity for SMSN detection, we analysed methylated 5′-CTGCAG sites in a native E. coli O104:H4 C227-11 strain35 (referred to as C227), and a matching WGA sample that is free of methylation (Fig. 2a; Methods). As expected, the sensitivity of the method increased with increasing covSM. For the SMSN approach, covSM≥15 enables detection of 6 mA with a sensitivity of 98.5% and a specificity of 99.5%. Although smaller library sizes can provide higher values of covSM, a minimum library size of 150–200 bp is recommended to avoid loss of genomic DNA during library construction and to facilitate removal of adapter-dimer constructs during purification.

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.