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1.
Opportunities and limitations of reduced representation bisulfite sequencing in plant ecological epigenomics.
Paun, O, Verhoeven, KJF, Richards, CL
The New phytologist. 2019;(2):738-742
Abstract
Contents Summary 738 I. Introduction 738 II. RRBS loci as genome-wide epigenetic markers 739 III. Exploiting functional annotation of RRBS loci 739 IV. Limitations of RRBS methods for nonmodel species 740 V. Maximising the impact of RRBS in plants 741 VI. Conclusions 741 Acknowledgements 741 SUMMARY Investigating the features and implications of epigenetic mechanisms across the breadth of organisms and ecosystems is important for understanding the ecological relevance of epigenetics. Several cost-effective reduced representation bisulfite sequencing approaches (RRBS) have been recently developed and applied to different organisms that lack a well annotated reference genome. These new approaches improve the assessment of epigenetic diversity in ecological settings and may provide functional insights. We assess here the opportunities and limitations of RRBS in nonmodel plant species. Well thought out experimental designs that include complementary gene expression studies, and the improvement of genomics resources for the target group, promise to maximize the effect of future RRBS studies.
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2.
DNA methylation detection: recent developments in bisulfite free electrochemical and optical approaches.
Bhattacharjee, R, Moriam, S, Umer, M, Nguyen, NT, Shiddiky, MJA
The Analyst. 2018;(20):4802-4818
Abstract
DNA methylation is one of the significant epigenetic modifications involved in mammalian development as well as in the initiation and progression of various diseases like cancer. Over the past few decades, an enormous amount of research has been carried out for the quantification of DNA methylation in the mammalian genome. Earlier, most of these methodologies used bisulfite treatment. However, the low conversion, false reading, longer assay time and complex chemical reaction are the common limitations of this method that hinder their application in routine clinical screening. Thus, as an alternative to bisulfite conversion-based DNA methylation detection, numerous bisulfite-free methods have been proposed. In this regard, electrochemical biosensors have gained much attention in recent years for being highly sensitive yet cost-effective, portable, and simple to operate. On the other hand, biosensors with optical readouts enable direct real time detection of biological molecules and are easily adaptable to multiplexing. Incorporation of electrochemical and optical readouts into bisulfite free DNA methylation analysis is paving the way for the translation of this important biomarker into standard patient care. In this review, we provide a critical overview of recent advances in the development of electrochemical and optical readout based bisulfite free DNA methylation assays.
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3.
Improvements in Genomic Technologies: Application to Crop Genomics.
Yuan, Y, Bayer, PE, Batley, J, Edwards, D
Trends in biotechnology. 2017;(6):547-558
Abstract
Second-generation sequencing (SGS) has advanced the study of crop genomes and has provided insights into diversity and evolution. However, repetitive DNA sequences in crops often lead to incomplete or erroneous assemblies because SGS reads are too short to fully resolve these repeats. To overcome some of these challenges, long-read sequencing and optical mapping have been developed to produce high-quality assemblies for complex genomes. Previously, high error rates, low throughput, and high costs have limited the adoption of long-read sequencing and optical mapping. However, with recent improvements and the development of novel algorithms, the application of these technologies is increasing. We review the development of long-read sequencing and optical mapping, and assess their application in crop genomics for breeding improved crops.
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4.
Strategies for analyzing bisulfite sequencing data.
Wreczycka, K, Gosdschan, A, Yusuf, D, Grüning, B, Assenov, Y, Akalin, A
Journal of biotechnology. 2017;:105-115
Abstract
DNA methylation is one of the main epigenetic modifications in the eukaryotic genome; it has been shown to play a role in cell-type specific regulation of gene expression, and therefore cell-type identity. Bisulfite sequencing is the gold-standard for measuring methylation over the genomes of interest. Here, we review several techniques used for the analysis of high-throughput bisulfite sequencing. We introduce specialized short-read alignment techniques as well as pre/post-alignment quality check methods to ensure data quality. Furthermore, we discuss subsequent analysis steps after alignment. We introduce various differential methylation methods and compare their performance using simulated and real bisulfite sequencing datasets. We also discuss the methods used to segment methylomes in order to pinpoint regulatory regions. We introduce annotation methods that can be used for further classification of regions returned by segmentation and differential methylation methods. Finally, we review software packages that implement strategies to efficiently deal with large bisulfite sequencing datasets locally and we discuss online analysis workflows that do not require any prior programming skills. The analysis strategies described in this review will guide researchers at any level to the best practices of bisulfite sequencing analysis.
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5.
Methods and novel technology for microRNA quantification in colorectal cancer screening.
Moody, L, He, H, Pan, YX, Chen, H
Clinical epigenetics. 2017;:119
Abstract
The screening and diagnosis of colorectal cancer (CRC) currently relies heavily on invasive endoscopic techniques as well as imaging and antigen detection tools. More accessible and reliable biomarkers are necessary for early detection in order to expedite treatment and improve patient outcomes. Recent studies have indicated that levels of specific microRNA (miRNA) are altered in CRC; however, measuring miRNA in biological samples has proven difficult, given the complicated and lengthy PCR-based procedures used by most laboratories. In this manuscript, we examine the potential of miRNA as CRC biomarkers, summarize the methods that have commonly been employed to quantify miRNA, and focus on novel strategies that can improve or replace existing technology for feasible implementation in a clinical setting. These include isothermal amplification techniques that can potentially eliminate the need for specialized thermocycling equipment. Additionally, we propose the use of near-infrared (NIR) probes which can minimize autofluorescence and photobleaching and streamline quantification without tedious sample processing. We suggest that novel miRNA quantification tools will be necessary to encourage new discoveries and facilitate their translation to clinical practice.
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6.
Lethal neonatal case and review of primary short-chain enoyl-CoA hydratase (SCEH) deficiency associated with secondary lymphocyte pyruvate dehydrogenase complex (PDC) deficiency.
Bedoyan, JK, Yang, SP, Ferdinandusse, S, Jack, RM, Miron, A, Grahame, G, DeBrosse, SD, Hoppel, CL, Kerr, DS, Wanders, RJA
Molecular genetics and metabolism. 2017;(4):342-349
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Abstract
Mutations in ECHS1 result in short-chain enoyl-CoA hydratase (SCEH) deficiency which mainly affects the catabolism of various amino acids, particularly valine. We describe a case compound heterozygous for ECHS1 mutations c.836T>C (novel) and c.8C>A identified by whole exome sequencing of proband and parents. SCEH deficiency was confirmed with very low SCEH activity in fibroblasts and nearly absent immunoreactivity of SCEH. The patient had a severe neonatal course with elevated blood and cerebrospinal fluid lactate and pyruvate concentrations, high plasma alanine and slightly low plasma cystine. 2-Methyl-2,3-dihydroxybutyric acid was markedly elevated as were metabolites of the three branched-chain α-ketoacids on urine organic acids analysis. These urine metabolites notably decreased when lactic acidosis decreased in blood. Lymphocyte pyruvate dehydrogenase complex (PDC) activity was deficient, but PDC and α-ketoglutarate dehydrogenase complex activities in cultured fibroblasts were normal. Oxidative phosphorylation analysis on intact digitonin-permeabilized fibroblasts was suggestive of slightly reduced PDC activity relative to control range in mitochondria. We reviewed 16 other cases with mutations in ECHS1 where PDC activity was also assayed in order to determine how common and generalized secondary PDC deficiency is associated with primary SCEH deficiency. For reasons that remain unexplained, we find that about half of cases with primary SCEH deficiency also exhibit secondary PDC deficiency. The patient died on day-of-life 39, prior to establishing his diagnosis, highlighting the importance of early and rapid neonatal diagnosis because of possible adverse effects of certain therapeutic interventions, such as administration of ketogenic diet, in this disorder. There is a need for better understanding of the pathogenic mechanisms and phenotypic variability in this relatively recently discovered disorder.
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Novel Strategies for Applied Metagenomics.
Moore-Connors, JM, Dunn, KA, Bielawski, JP, Van Limbergen, J
Inflammatory bowel diseases. 2016;(3):709-18
Abstract
Detailed analyses of the gut microbiome and its effect on human physiology and disease are emerging, thanks to advances in high-throughput DNA-sequencing technology and the burgeoning field of metagenomics. Metagenomics examines the structure and functional potential of microbial communities in their native habitats through the direct isolation and analysis of community DNA. In inflammatory bowel disease, gut microbiome studies have shown an association with perturbations in community composition and, especially, function. In this review, we discuss the application of next-generation sequencing to microbiome research and highlight the importance of modeling microbiome structure and function to the future of inflammatory bowel disease research and treatment.
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Development of molecular markers linked to disease resistance genes in common bean based on whole genome sequence.
Meziadi, C, Richard, MMS, Derquennes, A, Thareau, V, Blanchet, S, Gratias, A, Pflieger, S, Geffroy, V
Plant science : an international journal of experimental plant biology. 2016;:351-357
Abstract
Common bean (Phaseolus vulgaris) is the most important grain legume for direct human consumption in the world, particularly in developing countries where it constitutes the main source of protein. Unfortunately, common bean yield stability is constrained by a number of pests and diseases. As use of resistant genotypes is the most economic and ecologically safe means for controlling plant diseases, efforts have been made to genetically characterize resistance genes (R genes) in common bean. Despite its agronomic importance, genomic resources available in common bean were limited until the recent sequencing of common bean genome (Andean genotype G19833). Besides allowing the annotation of Nucleotide Binding-Leucine Rich Repeat (NB-LRR) encoding gene family, which is the prevalent class of disease R genes in plants, access to the whole genome sequence of common bean can be of great help for intense selection to increase the overall efficiency of crop improvement programs using marker-assisted selection (MAS). This review presents the state of the art of common bean NB-LRR gene clusters, their peculiar location in subtelomeres and correlation with genetically characterized monogenic R genes, as well as how the availability of the whole genome sequence can boost the development of molecular markers for MAS.
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9.
Next generation sequencing in endocrine practice.
Forlenza, GP, Calhoun, A, Beckman, KB, Halvorsen, T, Hamdoun, E, Zierhut, H, Sarafoglou, K, Polgreen, LE, Miller, BS, Nathan, B, et al
Molecular genetics and metabolism. 2015;(2-3):61-71
Abstract
With the completion of the Human Genome Project and advances in genomic sequencing technologies, the use of clinical molecular diagnostics has grown tremendously over the last decade. Next-generation sequencing (NGS) has overcome many of the practical roadblocks that had slowed the adoption of molecular testing for routine clinical diagnosis. In endocrinology, targeted NGS now complements biochemical testing and imaging studies. The goal of this review is to provide clinicians with a guide to the application of NGS to genetic testing for endocrine conditions, by compiling a list of established gene mutations detectable by NGS, and highlighting key phenotypic features of these disorders. As we outline in this review, the clinical utility of NGS-based molecular testing for endocrine disorders is very high. Identifying an exact genetic etiology improves understanding of the disease, provides clear explanation to families about the cause, and guides decisions about screening, prevention and/or treatment. To illustrate this approach, a case of hypophosphatasia with a pathogenic mutation in the ALPL gene detected by NGS is presented.
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10.
Methodological aspects of whole-genome bisulfite sequencing analysis.
Adusumalli, S, Mohd Omar, MF, Soong, R, Benoukraf, T
Briefings in bioinformatics. 2015;(3):369-79
Abstract
The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation beyond CpG sites and CpG islands. These technologies have opened new avenues to understand the interplay between epigenetic events, chromatin plasticity and gene regulation. However, the processing, managing and mining of this huge volume of data require specialized computational tools and statistical methods that are yet to be standardized. Here, we describe a complete bisulfite sequencing analysis workflow, including recently developed programs, highlighting each of the crucial analysis steps required, i.e. sequencing quality control, reads alignment, methylation scoring, methylation heterogeneity assessment, genomic features annotation, data visualization and determination of differentially methylated cytosines. Moreover, we discuss the limitations of these technologies and considerations to perform suitable analyses.