scholarly journals Mapping the epigenetic modifications of DNA and RNA

2020 ◽  
Vol 11 (11) ◽  
pp. 792-808 ◽  
Author(s):  
Lin-Yong Zhao ◽  
Jinghui Song ◽  
Yibin Liu ◽  
Chun-Xiao Song ◽  
Chengqi Yi
Keyword(s):  
Single Molecule ◽  
Detection Methods ◽  
Rna Modification ◽  
Rna Modifications ◽  
Single Molecule Sequencing ◽  
Dna And Rna ◽  
Third Generation Sequencing ◽  
Technological Advances ◽  
Long Read ◽  
High Throughput Detection

Abstract Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

scholarly journals Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 278
Author(s):  
Yuri Motorin ◽  
Virginie Marchand
Keyword(s):  
Single Molecule ◽  
Deep Sequencing ◽  
Rna Modification ◽  
Rna Modifications ◽  
Single Molecule Sequencing ◽  
Abasic Sites ◽  
Phosphate Chain ◽  
Adapter Ligation ◽  
Ribose Phosphate ◽  
Precise Mapping

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m6A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.

scholarly journals Recent advances in the detection of base modifications using the Nanopore sequencer

2019 ◽  
Vol 65 (1) ◽  
pp. 25-33 ◽  
Author(s):  
Liu Xu ◽  
Masahide Seki
Keyword(s):  
Single Molecule ◽  
Statistical Tests ◽  
Regulation Of Gene Expression ◽  
Detection Methods ◽  
Open Chromatin ◽  
Rna Molecules ◽  
Base Modification ◽  
Dna And Rna ◽  
Long Read ◽  
Nanopore Sequencer

Abstract DNA and RNA modifications have important functions, including the regulation of gene expression. Existing methods based on short-read sequencing for the detection of modifications show difficulty in determining the modification patterns of single chromosomes or an entire transcript sequence. Furthermore, the kinds of modifications for which detection methods are available are very limited. The Nanopore sequencer is a single-molecule, long-read sequencer that can directly sequence RNA as well as DNA. Moreover, the Nanopore sequencer detects modifications on long DNA and RNA molecules. In this review, we mainly focus on base modification detection in the DNA and RNA of mammals using the Nanopore sequencer. We summarize current studies of modifications using the Nanopore sequencer, detection tools using statistical tests or machine learning, and applications of this technology, such as analyses of open chromatin, DNA replication, and RNA metabolism.

scholarly journals Error correction and assembly complexity of single molecule sequencing reads.

2014 ◽  
Author(s):  
Hayan Lee ◽  
James Gurtowski ◽  
Shinjae Yoo ◽  
Shoshana Marcus ◽  
W. Richard McCombie ◽  
...  
Keyword(s):  
Error Correction ◽  
Single Molecule ◽  
Support Vector ◽  
Assembly Model ◽  
Single Molecule Sequencing ◽  
Dna And Rna ◽  
Long Read ◽  
Error Correction Algorithm ◽  
Assembly Performance ◽  
The Impact

Third generation single molecule sequencing technology is poised to revolutionize genomics by enabling the sequencing of long, individual molecules of DNA and RNA. These technologies now routinely produce reads exceeding 5,000 basepairs, and can achieve reads as long as 50,000 basepairs. Here we evaluate the limits of single molecule sequencing by assessing the impact of long read sequencing in the assembly of the human genome and 25 other important genomes across the tree of life. From this, we develop a new data-driven model using support vector regression that can accurately predict assembly performance. We also present a novel hybrid error correction algorithm for long PacBio sequencing reads that uses pre-assembled Illumina sequences for the error correction. We apply it several prokaryotic and eukaryotic genomes, and show it can achieve near-perfect assemblies of small genomes (< 100Mbp) and substantially improved assemblies of larger ones. All source code and the assembly model are available open-source.

scholarly journals Long-range single-molecule mapping of chromatin modification in eukaryotes

2021 ◽  
Author(s):  
Zhe Weng ◽  
Fengying Ruan ◽  
Weitian Chen ◽  
Zhe Xie ◽  
Yeming Xie ◽  
...  
Keyword(s):  
Histone Modification ◽  
Long Range ◽  
Single Molecule ◽  
Protein A ◽  
Chromatin Modification ◽  
Protein Binding Sites ◽  
Short Read Sequencing ◽  
Third Generation Sequencing ◽  
Long Read ◽  
Generation Sequencing

The epigenetic modifications of histones are essential marks related to the development and disease pathogenesis, including human cancers. Mapping histone modification has emerged as the widely used tool for studying epigenetic regulation. However, existing approaches limited by fragmentation and short-read sequencing cannot provide information about the long-range chromatin states and represent the average chromatin status in samples. We leveraged the advantage of long read sequencing to develop a method "BIND&MODIFY" for profiling the histone modification of individual DNA fiber. Our approach is based on the recombinant fused protein A-EcoGII, which tethers the methyltransferase EcoGII to the protein binding sites and locally labels the neighboring DNA regions through artificial methylations. We demonstrate that the aggregated BIND&MODIFY signal matches the bulk-level ChIP-seq and CUT&TAG, observe the single-molecule heterogenous histone modification status, and quantify the correlation between distal elements. This method could be an essential tool in the future third-generation sequencing ages.

scholarly journals Emerging approaches for detection of methylation sites in RNA

Open Biology ◽  
2018 ◽  
Vol 8 (9) ◽  
pp. 180121 ◽  
Author(s):  
Anna Ovcharenko ◽  
Andrea Rentmeister
Keyword(s):  
High Throughput Sequencing ◽  
De Novo ◽  
Direct Detection ◽  
False Negative ◽  
Detection Methods ◽  
Rna Modifications ◽  
Negative Results ◽  
Third Generation Sequencing ◽  
False Negative Results ◽  
Generation Sequencing

RNA methylations play a significant regulatory role in diverse biological processes. Although the transcriptome-wide discovery of unknown RNA methylation sites is essential to elucidate their function, the development of a bigger variety of detection approaches is desirable for multiple reasons. Many established detection methods for RNA modifications heavily rely on the specificity of the respective antibodies. Thus, the development of antibody-independent transcriptome-wide methods is beneficial. Even the antibody-independent high-throughput sequencing-based methods are liable to produce false-positive or false-negative results. The development of an independent method for each modification could help validate the detected modification sites. Apart from the transcriptome-wide methods for methylation detection de novo , methods for monitoring the presence of a single methylation at a determined site are also needed. In contrast to the transcriptome-wide detection methods, the techniques used for monitoring purposes need to be cheap, fast and easy to perform. This review considers modern approaches for site-specific detection of methylated nucleotides in RNA. We also discuss the potential of third-generation sequencing methods for direct detection of RNA methylations.

scholarly journals Contig annotation tool CAT robustly classifies assembled metagenomic contigs and long sequences

2016 ◽  
Author(s):  
Diego D. Cambuy ◽  
Felipe H. Coutinho ◽  
Bas E. Dutilh
Keyword(s):  
Single Molecule ◽  
Dna Sequences ◽  
Taxonomic Classification ◽  
Annotation Tool ◽  
Single Molecule Sequencing ◽  
Short Read ◽  
Long Read ◽  
Micro Organisms ◽  
Taxonomic Annotation

AbstractIn modern-day metagenomics, there is an increasing need for robust taxonomic annotation of long DNA sequences from unknown micro-organisms. Long metagenomic sequences may be derived from assembly of short-read metagenomes, or from long-read single molecule sequencing. Here we introduce CAT, a pipeline for robust taxonomic classification of long DNA sequences. We show that CAT correctly classifies contigs at different taxonomic levels, even in simulated metagenomic datasets that are very distantly related from the sequences in the database. CAT is implemented in Python and the required scripts can be freely downloaded from Github.

scholarly journals Haplotype Diversity and Sequence Heterogeneity of Human Telomeres

2020 ◽  
Author(s):  
Kirill Grigorev ◽  
Jonathan Foox ◽  
Daniela Bezdan ◽  
Daniel Butler ◽  
Jared J. Luxton ◽  
...  
Keyword(s):  
Single Molecule ◽  
Disease Risk ◽  
Haplotype Diversity ◽  
Single Molecule Sequencing ◽  
Sequence Heterogeneity ◽  
Repetitive Nature ◽  
Telomere Sequence ◽  
Long Read ◽  
Motif Composition ◽  
Intractable Problem

AbstractTelomeres are regions of repetitive nucleotide sequences capping the ends of eukaryotic chromosomes that protect against deterioration, whose lengths can be correlated with age and disease risk factors. Given their length and repetitive nature, telomeric regions are not easily reconstructed from short read sequencing, making telomere sequence resolution a very costly and generally intractable problem. Recently, long-read sequencing, with read lengths measuring in hundreds of Kbp, has made it possible to routinely read into telomeric regions and inspect their structure. Here, we describe a framework for extracting telomeric reads from single-molecule sequencing experiments, describing their sequence variation and motifs, and for haplotype inference. We find that long telomeric stretches can be accurately captured with long-read sequencing, observe extensive sequence heterogeneity of human telomeres, discover and localize non-canonical motifs (both previously reported as well as novel), and report the first motif composition maps of human telomeric diplotypes on a multi-Kbp scale.

scholarly journals A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production.

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 961
Author(s):  
Kevin McKernan ◽  
Liam Kane ◽  
Yvonne Helbert ◽  
Lei Zhang ◽  
Nathan Houde ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Single Molecule ◽  
Reference Genome ◽  
De Novo ◽  
Genomic Diversity ◽  
Sequence Coverage ◽  
Single Molecule Sequencing ◽  
Contiguous Gene ◽  
Long Read ◽  
Interesting Variation

The Psilocybe genus is well known for the synthesis of valuable psychoactive compounds such as Psilocybin, Psilocin, Baeocystin and Aeruginascin. The ubiquity of Psilocybin synthesis in Psilocybe has been attributed to a horizontal gene transfer mechanism of a ~20Kb gene cluster. A recently published highly contiguous reference genome derived from long read single molecule sequencing has underscored interesting variation in this Psilocybin synthesis gene cluster. This reference genome has also enabled the shotgun sequencing of spores from many Psilocybe strains to better catalog the genomic diversity in the Psilocybin synthesis pathway. Here we present the de novo assembly of 81 Psilocybe genomes compared to the P.envy reference genome. Surprisingly, the genomes of Psilocybe galindoi, Psilocybe tampanensis and Psilocybe azurescens lack sequence coverage over the previously described Psilocybin synthesis pathway but do demonstrate amino acid sequence homology to a less contiguous gene cluster and may illuminate the previously proposed evolution of psilocybin synthesis.

scholarly journals AsmMix: A pipeline for high quality diploid de novo assembly

2021 ◽  
Author(s):  
Pei Wu ◽  
Chao Liu ◽  
Ou Wang ◽  
Xia Zhao ◽  
Fang Chen ◽  
...  
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Variant Calling ◽  
The Other ◽  
Second Step ◽  
Small Scale ◽  
Mixing Process ◽  
High Quality ◽  
Single Molecule Sequencing ◽  
Long Read

AbstractIn this paper, we report a pipeline, AsmMix, which is capable of producing both contiguous and high-quality diploid genomes. The pipeline consists of two steps. In the first step, two sets of assemblies are generated: one is based on co-barcoded reads, which are highly accurate and haplotype-resolved but contain many gaps, the other assembly is based on single-molecule sequencing reads, which is contiguous but error-prone. In the second step, those two sets of assemblies are compared and integrated into a haplotype-resolved assembly with fewer errors. We test our pipeline using a dataset of human genome NA24385, perform variant calling from those assemblies and then compare against GIAB Benchmark. We show that AsmMix pipeline could produce highly contiguous, accurate, and haplotype-resolved assemblies. Especially the assembly mixing process could effectively reduce small-scale errors in the long read assembly.

scholarly journals Detecting AGG Interruptions in Females With a FMR1 Premutation by Long-Read Single-Molecule Sequencing: A 1 Year Clinical Experience

2018 ◽  
Vol 9 ◽  
Author(s):  
Simon Ardui ◽  
Valerie Race ◽  
Thomy de Ravel ◽  
Hilde Van Esch ◽  
Koenraad Devriendt ◽  
...  
Keyword(s):  
Clinical Experience ◽  
Single Molecule ◽  
Single Molecule Sequencing ◽  
Fmr1 Premutation ◽  
Long Read ◽  
Agg Interruptions
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