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1.
Entailing the Next-Generation Sequencing and Metabolome for Sustainable Agriculture by Improving Plant Tolerance.
Ashraf, MF, Hou, D, Hussain, Q, Imran, M, Pei, J, Ali, M, Shehzad, A, Anwar, M, Noman, A, Waseem, M, et al
International journal of molecular sciences. 2022;(2)
Abstract
Crop production is a serious challenge to provide food for the 10 billion individuals forecasted to live across the globe in 2050. The scientists' emphasize establishing an equilibrium among diversity and quality of crops by enhancing yield to fulfill the increasing demand for food supply sustainably. The exploitation of genetic resources using genomics and metabolomics strategies can help generate resilient plants against stressors in the future. The innovation of the next-generation sequencing (NGS) strategies laid the foundation to unveil various plants' genetic potential and help us to understand the domestication process to unmask the genetic potential among wild-type plants to utilize for crop improvement. Nowadays, NGS is generating massive genomic resources using wild-type and domesticated plants grown under normal and harsh environments to explore the stress regulatory factors and determine the key metabolites. Improved food nutritional value is also the key to eradicating malnutrition problems around the globe, which could be attained by employing the knowledge gained through NGS and metabolomics to achieve suitability in crop yield. Advanced technologies can further enhance our understanding in defining the strategy to obtain a specific phenotype of a crop. Integration among bioinformatic tools and molecular techniques, such as marker-assisted, QTLs mapping, creation of reference genome, de novo genome assembly, pan- and/or super-pan-genomes, etc., will boost breeding programs. The current article provides sequential progress in NGS technologies, a broad application of NGS, enhancement of genetic manipulation resources, and understanding the crop response to stress by producing plant metabolites. The NGS and metabolomics utilization in generating stress-tolerant plants/crops without deteriorating a natural ecosystem is considered a sustainable way to improve agriculture production. This highlighted knowledge also provides useful research that explores the suitable resources for agriculture sustainability.
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2.
Novel GRHL2 Gene Variant Associated with Hearing Loss: A Case Report and Review of the Literature.
Trebusak Podkrajsek, K, Tesovnik, T, Bozanic Urbancic, N, Battelino, S
Genes. 2021;(4)
Abstract
In contrast to the recessive form, hearing loss inherited in a dominant manner is more often post-lingual and typically results in a progressive sensorineural hearing loss with variable severity and late onset. Variants in the GRHL2 gene are an extremely rare cause of dominantly inherited hearing loss. Genetic testing is a crucial part of the identification of the etiology of hearing loss in individual patients, especially when performed with next-generation sequencing, enabling simultaneous analysis of numerous genes, including those rarely associated with hearing loss. We aimed to evaluate the genetic etiology of hearing loss in a family with moderate late-onset hearing loss using next-generation sequencing and to conduct a review of reported variants in the GRHL2 gene. We identified a novel disease-causing variant in the GRHL2 gene (NM_024915: c.1510C>T; p.Arg504Ter) in both affected members of the family. They both presented with moderate late-onset hearing loss with no additional clinical characteristics. Reviewing known GRHL2 variants associated with hearing loss, we can conclude that they are more likely to be truncating variants, while the associated onset of hearing loss is variable.
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3.
The Methods of Digging for "Gold" within the Salt: Characterization of Halophilic Prokaryotes and Identification of Their Valuable Biological Products Using Sequencing and Genome Mining Tools.
Lach, J, Jęcz, P, Strapagiel, D, Matera-Witkiewicz, A, Stączek, P
Genes. 2021;(11)
Abstract
Halophiles, the salt-loving organisms, have been investigated for at least a hundred years. They are found in all three domains of life, namely Archaea, Bacteria, and Eukarya, and occur in saline and hypersaline environments worldwide. They are already a valuable source of various biomolecules for biotechnological, pharmaceutical, cosmetological and industrial applications. In the present era of multidrug-resistant bacteria, cancer expansion, and extreme environmental pollution, the demand for new, effective compounds is higher and more urgent than ever before. Thus, the unique metabolism of halophilic microorganisms, their low nutritional requirements and their ability to adapt to harsh conditions (high salinity, high pressure and UV radiation, low oxygen concentration, hydrophobic conditions, extreme temperatures and pH, toxic compounds and heavy metals) make them promising candidates as a fruitful source of bioactive compounds. The main aim of this review is to highlight the nucleic acid sequencing experimental strategies used in halophile studies in concert with the presentation of recent examples of bioproducts and functions discovered in silico in the halophile's genomes. We point out methodological gaps and solutions based on in silico methods that are helpful in the identification of valuable bioproducts synthesized by halophiles. We also show the potential of an increasing number of publicly available genomic and metagenomic data for halophilic organisms that can be analysed to identify such new bioproducts and their producers.
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4.
The Battle to Sequence the Bread Wheat Genome: A Tale of the Three Kingdoms.
Guan, J, Garcia, DF, Zhou, Y, Appels, R, Li, A, Mao, L
Genomics, proteomics & bioinformatics. 2020;(3):221-229
Abstract
In the year 2018, the world witnessed the finale of the race to sequence the genome of the world's most widely grown crop, the common wheat. Wheat has been known to bear a notoriously large and complicated genome of a polyploidy nature. A decade competition to sequence the wheat genome initiated with a single consortium of multiple countries, taking a conventional strategy similar to that for sequencing Arabidopsis and rice, became ferocious over time as both sequencing technologies and genome assembling methodologies advanced. At different stages, multiple versions of genome sequences of the same variety (e.g., Chinese Spring) were produced by several groups with their special strategies. Finally, 16 years after the rice genome was finished and 9 years after that of maize, the wheat research community now possesses its own reference genome. Armed with these genomics tools, wheat will reestablish itself as a model for polyploid plants in studying the mechanisms of polyploidy evolution, domestication, genetic and epigenetic regulation of homoeolog expression, as well as defining its genetic diversity and breeding on the genome level. The enhanced resolution of the wheat genome should also help accelerate development of wheat cultivars that are more tolerant to biotic and/or abiotic stresses with better quality and higher yield.
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5.
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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6.
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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7.
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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8.
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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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.
Fundamentals of pyrosequencing.
Harrington, CT, Lin, EI, Olson, MT, Eshleman, JR
Archives of pathology & laboratory medicine. 2013;(9):1296-303
Abstract
CONTEXT DNA sequencing is critical to identifying many human genetic disorders caused by DNA mutations, including cancer. Pyrosequencing is less complex, involves fewer steps, and has a superior limit of detection compared with Sanger sequencing. The fundamental basis of pyrosequencing is that pyrophosphate is released when a deoxyribonucleotide triphosphate is added to the end of a nascent strand of DNA. Because deoxyribonucleotide triphosphates are sequentially added to the reaction and because the pyrophosphate concentration is continuously monitored, the DNA sequence can be determined. OBJECTIVE To demonstrate the fundamental principles of pyrosequencing. DATA SOURCES Salient features of pyrosequencing are demonstrated using the free software program Pyromaker ( http://pyromaker.pathology.jhmi.edu ), through which users can input DNA sequences and other pyrosequencing parameters to generate the expected pyrosequencing results. CONCLUSIONS We demonstrate how mutant and wild-type DNA sequences result in different pyrograms. Using pyrograms of established mutations in tumors, we explain how to analyze the pyrogram peaks generated by different dispensation sequences. Further, we demonstrate some limitations of pyrosequencing, including how some complex mutations can be indistinguishable from single base mutations. Pyrosequencing is the basis of the Roche 454 next-generation sequencer and many of the same principles also apply to the Ion Torrent hydrogen ion-based next-generation sequencers.