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

Comprehensive genome-wide analyses of bacterial DNA methylation have not been possible until the recent advent of single molecule, real-time (SMRT) sequencing. This technology enables the direct detection of N6-methyladenine (6mA) and 4-methylcytosine (4mC) at single nucleotide resolution on a genome-wide scale. The distributions of these two major types of DNA methylation, along with 5-methylcytosine (5mC), comprise the bacterial methylome, some rare exceptions notwithstanding. SMRT sequencing has already revealed marked diversity in bacterial methylomes as well as the existence of heterogeneity of methylation in cells in single bacterial colonies, where such ‘epigenetic’ variation can enable bacterial populations to rapidly adapt to changing conditions. However, current methods for studying bacterial methylomes using SMRT sequencing mainly rely on population-level summaries that do not provide the single-cell resolution necessary for dissecting the epigenetic heterogeneity in bacterial populations. Here, we present a novel SMRT sequencing-based framework, consisting of two complementary methods, for single molecule-level detection of DNA methylation and assessment of methyltransferase activity through single molecule-level long read-based epigenetic phasing. Using seven bacterial strains and integrating data from SMRT and Illumina sequencing, we show that our method yields significantly improved resolution compared to existing population-level methods, and reveals several distinct types of epigenetic heterogeneity. Our approach enables new investigations of the complex architecture and dynamics of bacterial methylomes and provides a powerful new tool for the study of bacterial epigenetic control.

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Analysis of Transcripts and splice isoforms in Red Clover (Trifolium pratense L.) by single-molecule long-read sequencing

10.1101/330977 ◽

2018 ◽

Author(s):

Yuehui Chao ◽

Jianbo Yuan ◽

Sifeng Li ◽

Siqiao Jia ◽

Liebao Han ◽

...

Keyword(s):

Single Molecule ◽

Trifolium Pratense ◽

Intron Retention ◽

Draft Genome ◽

Red Clover ◽

Full Length ◽

Splice Isoforms ◽

Single Molecule Level ◽

Long Read ◽

Novel Isoforms

AbstractRed clover (Trifolium pratense L.) is an important cool-season legume plant, which is the most widely planted forage legume after alfalfa. Although a draft genome sequence was published already, the sequences and completed structure of mRNA transcripts remain unclear, which limit further explore on red clover. In this study, the red clover transcriptome was sequenced using single-molecule long-read sequencing to identify full-length splice isoforms, and 29,730 novel isoforms from known genes and 2,194 novel isoforms from novel genes were identified. A total of 5,492 alternative splicing events was identified and the majority of alter spliced events in red clover was corrected as intron retention. In addition, of the 15,229 genes detected by SMRT, 8,719 including 1,86,517 transcripts have at least one poly(A) site. Furthermore, we identified 4,333 long non-coding RNAs and 3,762 fusion transcripts. Our results show the feasibility of deep sequencing full-length RNA from red clover transcriptome on a single-molecule level.

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Minimal detection and low biological fluctuation of mitochondrial CpG methylation at the single-molecule level

10.1101/2020.09.14.296269 ◽

2020 ◽

Author(s):

Chloe Goldsmith ◽

Jesús Rafael Rodríguez-Aguilera ◽

Ines El-Rifai ◽

Adrien Jarretier ◽

Valérie Hervieu ◽

...

Keyword(s):

Cell Lines ◽

Single Molecule ◽

Nuclear Dna ◽

Mitochondrial Genes ◽

Cpg Methylation ◽

Nanopore Sequencing ◽

Biological Processes ◽

Single Molecule Level ◽

Long Read ◽

Low Levels

AbstractCytosine DNA methylation in the CpG context (5mCpG) is associated with the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mCpG has a regulatory role in this context. The main aim of this work was to develop and validate a novel tool for determining methylation of mtDNA and to corroborate its existence across different biological contexts. Using long-read nanopore sequencing we found low levels of CpG methylation (with few exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG was overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Although we do show several significant changes in all these conditions, 5mCpG is unlikely to play a major role in defining the transcriptional status of mitochondrial genes.

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DiMeLo-seq: Directed Methylation with Long-read sequencing v2

10.17504/protocols.io.b2u8qezw ◽

2021 ◽

Author(s):

Nicolas Altemose ◽

Annie Maslan ◽

Owen Smith ◽

Kousik Sundararajan ◽

Rachel Brown ◽

...

Keyword(s):

Single Molecule ◽

Protein Interactions ◽

Pcr Amplification ◽

Dna Interaction ◽

Dna Interactions ◽

Single Molecule Level ◽

Multiple Protein ◽

Protein Dna Interactions ◽

Adenine Methylation ◽

Long Read

Directed Methylation and Long-read sequencing (DiMeLo-seq) is a powerful method to map protein-DNA interactions at a single-molecule level across the genome (including repetitive regions). It can be multiplexed to analyze multiple base modifications at once (e.g. endogenous CpG methylation and directed pA-Hia5 adenine methylation). Additionally, PCR amplification is not necessary for this protocol, which means that sequencing readout is proportional to protein-DNA interaction frequency. Finally, DiMeLo-seq can be used to map multiple protein interactions across a long single molecule.

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Low biological fluctuation of mitochondrial CpG and non-CpG methylation at the single-molecule level

Scientific Reports ◽

10.1038/s41598-021-87457-8 ◽

2021 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Chloe Goldsmith ◽

Jesús Rafael Rodríguez-Aguilera ◽

Ines El-Rifai ◽

Adrien Jarretier-Yuste ◽

Valérie Hervieu ◽

...

Keyword(s):

Cell Lines ◽

Single Molecule ◽

Nuclear Dna ◽

Pcr Amplification ◽

Nanopore Sequencing ◽

High Coverage ◽

Single Molecule Level ◽

Modified Dna ◽

Long Read ◽

Low Levels

AbstractMammalian cytosine DNA methylation (5mC) is associated with the integrity of the genome and the transcriptional status of nuclear DNA. Due to technical limitations, it has been less clear if mitochondrial DNA (mtDNA) is methylated and whether 5mC has a regulatory role in this context. Here, we used bisulfite-independent single-molecule sequencing of native human and mouse DNA to study mitochondrial 5mC across different biological conditions. We first validated the ability of long-read nanopore sequencing to detect 5mC in CpG (5mCpG) and non-CpG (5mCpH) context in nuclear DNA at expected genomic locations (i.e. promoters, gene bodies, enhancers, and cell type-specific transcription factor binding sites). Next, using high coverage nanopore sequencing we found low levels of mtDNA CpG and CpH methylation (with several exceptions) and little variation across biological processes: differentiation, oxidative stress, and cancer. 5mCpG and 5mCpH were overall higher in tissues compared to cell lines, with small additional variation between cell lines of different origin. Despite general low levels, global and single-base differences were found in cancer tissues compared to their adjacent counterparts, in particular for 5mCpG. In conclusion, nanopore sequencing is a useful tool for the detection of modified DNA bases on mitochondria that avoid the biases introduced by bisulfite and PCR amplification. Enhanced nanopore basecalling models will provide further resolution on the small size effects detected here, as well as rule out the presence of other DNA modifications such as oxidized forms of 5mC.

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Faculty Opinions recommendation of Two-substrate association with the 20S proteasome at single-molecule level.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020051.231447 ◽

2004 ◽

Author(s):

Jane Endicott

Keyword(s):

Single Molecule ◽

20S Proteasome ◽

Single Molecule Level

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Faculty Opinions recommendation of Stochastic protein expression in individual cells at the single molecule level.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1032296.372996 ◽

2006 ◽

Author(s):

Ulf Pettersson

Keyword(s):

Protein Expression ◽

Single Molecule ◽

Single Molecule Level

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Atomic Force Microscopy (AFM) for Topography and Recognition Imaging at Single Molecule Level

Encyclopedia of Biophysics ◽

10.1007/978-3-642-16712-6_496 ◽

2013 ◽

pp. 102-112

Author(s):

Memed Duman ◽

Andreas Ebner ◽

Christian Rankl ◽

Jilin Tang ◽

Lilia A. Chtcheglova ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Single Molecule ◽

Single Molecule Level ◽

Force Microscopy ◽

Atomic Force ◽

Recognition Imaging

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Analysis of HLA-G long-read genomic sequences in mother–offspring pairs with preeclampsia

Scientific Reports ◽

10.1038/s41598-020-77081-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ayako Nishizawa ◽

Kazuki Kumada ◽

Keiko Tateno ◽

Maiko Wagata ◽

Sakae Saito ◽

...

Keyword(s):

Single Molecule ◽

Gene Polymorphisms ◽

Genomic Dna ◽

Genomic Sequences ◽

Genomic Sequencing ◽

Public Database ◽

Coding Sequences ◽

Pacbio Rs Ii ◽

Potential Association ◽

Long Read

AbstractPreeclampsia is a pregnancy-induced disorder that is characterized by hypertension and is a leading cause of perinatal and maternal–fetal morbidity and mortality. HLA-G is thought to play important roles in maternal–fetal immune tolerance, and the associations between HLA-G gene polymorphisms and the onset of pregnancy-related diseases have been explored extensively. Because contiguous genomic sequencing is difficult, the association between the HLA-G genotype and preeclampsia onset is controversial. In this study, genomic sequences of the HLA-G region (5.2 kb) from 31 pairs of mother–offspring genomic DNA samples (18 pairs from normal pregnancies/births and 13 from preeclampsia births) were obtained by single-molecule real-time sequencing using the PacBio RS II platform. The HLA-G alleles identified in our cohort matched seven known HLA-G alleles, but we also identified two new HLA-G alleles at the fourth-field resolution and compared them with nucleotide sequences from a public database that consisted of coding sequences that cover the 3.1-kb HLA-G gene span. Intriguingly, a potential association between preeclampsia onset and the poly T stretch within the downstream region of the HLA-G*01:01:01:01 allele was found. Our study suggests that long-read sequencing of HLA-G will provide clues for characterizing HLA-G variants that are involved in the pathophysiology of preeclampsia.

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