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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.


SMALR methods for methylation detection in SMRT reads.Schematic illustrating the general approaches of both the existing and two proposed SMALR methods for detecting DNA methylation in SMRT sequencing reads. (a) A single SMRT sequencing molecule (short DNA insert+adapters) and the subreads that are produced during sequencing. (b) The existing methylation detection method is based on a molecule-aggregated, single-nucleotide (AggSN) score. For a given strand and genomic position, the IPD values from all the subreads aligning to that strand and position are aggregated together across all molecules to infer the presence of a consensus methylated base. (c) The proposed single molecule, single nucleotide (SMSN) method for detecting DNA methylation relies instead on separate consideration of subreads from different molecules. The SMSN scores are calculated for each molecule, strand and genomic position. (d) A single SMRT sequencing molecule (long DNA insert+adapters) with a single long subread and the proposed single molecule, pooled (SMP) approach for assessing MTase activity. This approach pools together IPD values from multiple motif sites along the length of a single long subread.
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f1: SMALR methods for methylation detection in SMRT reads.Schematic illustrating the general approaches of both the existing and two proposed SMALR methods for detecting DNA methylation in SMRT sequencing reads. (a) A single SMRT sequencing molecule (short DNA insert+adapters) and the subreads that are produced during sequencing. (b) The existing methylation detection method is based on a molecule-aggregated, single-nucleotide (AggSN) score. For a given strand and genomic position, the IPD values from all the subreads aligning to that strand and position are aggregated together across all molecules to infer the presence of a consensus methylated base. (c) The proposed single molecule, single nucleotide (SMSN) method for detecting DNA methylation relies instead on separate consideration of subreads from different molecules. The SMSN scores are calculated for each molecule, strand and genomic position. (d) A single SMRT sequencing molecule (long DNA insert+adapters) with a single long subread and the proposed single molecule, pooled (SMP) approach for assessing MTase activity. This approach pools together IPD values from multiple motif sites along the length of a single long subread.

Mentions: Many studies have used SMRT sequencing to conduct de novo identification of MTase target motifs, revealing surprising diversity in motifs among bacterial species and even among closely related strains353637383940. However, the heterogeneities within bacterial populations cultured under different conditions have not been thoroughly explored. This is primarily limited by the current SMRT sequencing-based bacterial methylome analysis protocol, which relies on assessments of aggregate IPD values at each genomic position across populations of cells33343541; the individual reads (each from a single DNA molecule) are aligned to the same genomic region and then statistically modelled as an ensemble (Fig. 1a,b). This approach enhances the statistical power for methylation detection at single-nucleotide resolution, but fundamentally limits the ability to resolve epigenetic heterogeneity within the sample. Although progress beyond traditional population-level analysis has recently been reported3842, new methods are needed to provide improved resolution on complex methylomes.


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)

SMALR methods for methylation detection in SMRT reads.Schematic illustrating the general approaches of both the existing and two proposed SMALR methods for detecting DNA methylation in SMRT sequencing reads. (a) A single SMRT sequencing molecule (short DNA insert+adapters) and the subreads that are produced during sequencing. (b) The existing methylation detection method is based on a molecule-aggregated, single-nucleotide (AggSN) score. For a given strand and genomic position, the IPD values from all the subreads aligning to that strand and position are aggregated together across all molecules to infer the presence of a consensus methylated base. (c) The proposed single molecule, single nucleotide (SMSN) method for detecting DNA methylation relies instead on separate consideration of subreads from different molecules. The SMSN scores are calculated for each molecule, strand and genomic position. (d) A single SMRT sequencing molecule (long DNA insert+adapters) with a single long subread and the proposed single molecule, pooled (SMP) approach for assessing MTase activity. This approach pools together IPD values from multiple motif sites along the length of a single long subread.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: SMALR methods for methylation detection in SMRT reads.Schematic illustrating the general approaches of both the existing and two proposed SMALR methods for detecting DNA methylation in SMRT sequencing reads. (a) A single SMRT sequencing molecule (short DNA insert+adapters) and the subreads that are produced during sequencing. (b) The existing methylation detection method is based on a molecule-aggregated, single-nucleotide (AggSN) score. For a given strand and genomic position, the IPD values from all the subreads aligning to that strand and position are aggregated together across all molecules to infer the presence of a consensus methylated base. (c) The proposed single molecule, single nucleotide (SMSN) method for detecting DNA methylation relies instead on separate consideration of subreads from different molecules. The SMSN scores are calculated for each molecule, strand and genomic position. (d) A single SMRT sequencing molecule (long DNA insert+adapters) with a single long subread and the proposed single molecule, pooled (SMP) approach for assessing MTase activity. This approach pools together IPD values from multiple motif sites along the length of a single long subread.
Mentions: Many studies have used SMRT sequencing to conduct de novo identification of MTase target motifs, revealing surprising diversity in motifs among bacterial species and even among closely related strains353637383940. However, the heterogeneities within bacterial populations cultured under different conditions have not been thoroughly explored. This is primarily limited by the current SMRT sequencing-based bacterial methylome analysis protocol, which relies on assessments of aggregate IPD values at each genomic position across populations of cells33343541; the individual reads (each from a single DNA molecule) are aligned to the same genomic region and then statistically modelled as an ensemble (Fig. 1a,b). This approach enhances the statistical power for methylation detection at single-nucleotide resolution, but fundamentally limits the ability to resolve epigenetic heterogeneity within the sample. Although progress beyond traditional population-level analysis has recently been reported3842, new methods are needed to provide improved resolution on complex methylomes.

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.