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The next generation sequencing enables generation of high resolution and high throughput data for structure sequence of any genome at a fast declining cost. This opens opportunity for population based genetic and genomic analyses. In many applications, whole genome sequencing or re-sequencing is unnecessary or prohibited by budget limits. The Reduced Representation Genome Sequencing (RRGS), which sequences only a small proportion of the genome of interest, has been proposed to deal with the situations. Several forms of RRGS are proposed and implemented in the literature. When applied to plant or crop species, the current RRGS protocols shared a key drawback that a significantly high proportion (up to 60%) of sequence reads to be generated may be of non-genomic origin but attributed to chloroplast DNA or rRNA genes, leaving an exceptional low efficiency of the sequencing experiment. We recommended and discussed here the design of optimized simplified genomic DNA and bisulfite sequencing strategies, which may greatly improves efficiency of the sequencing experiments by bringing down the presentation of the undesirable sequencing reads to less than 10% in the whole sequence reads. The optimized RAD-seq and RRBS-seq methods are potentially useful for sequence variant screening and genotyping in large plant/crop populations. 相似文献
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Jamie R. Weaver Marisa S. Bartolomei 《Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms》2014,1839(3):169-177
Genomic imprinting is an epigenetic phenomenon in which genes are expressed monoallelically in a parent-of-origin-specific manner. Each chromosome is imprinted with its parental identity. Here we will discuss the nature of this imprinting mark. DNA methylation has a well-established central role in imprinting, and the details of DNA methylation dynamics and the mechanisms that target it to imprinted loci are areas of active investigation. However, there is increasing evidence that DNA methylation is not solely responsible for imprinted expression. At the same time, there is growing appreciation for the contributions of post-translational histone modifications to the regulation of imprinting. The integration of our understanding of these two mechanisms is an important goal for the future of the imprinting field. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development. 相似文献
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《Epigenetics》2013,8(11):1540-1556
Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ~3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ~2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ~750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain. 相似文献
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Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain. 相似文献
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DNA microarray and next-generation DNA sequencing technologies are important tools for high-throughput genome research, in
revealing both the structural and functional characteristics of genomes. In the past decade the DNA microarray technologies
have been widely applied in the studies of functional genomics, systems biology and pharmacogenomics. The next-generation
DNA sequencing method was first introduced by the 454 Company in 2003, immediately followed by the establishment of the Solexa
and Solid techniques by other biotech companies. Though it has not been long since the first emergence of this technology,
with the fast and impressive improvement, the application of this technology has extended to almost all fields of genomics
research, as a rival challenging the existing DNA microarray technology. This paper briefly reviews the working principles
of these two technologies as well as their application and perspectives in genome research.
Supported by the National High-Tech Research Program of China (Grant No.2006AA020704) and Shanghai Science and Technology
Commission (Grant No. 05DZ22201) 相似文献
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Tom Oosting Elena Hilario Maren Wellenreuther Peter A. Ritchie 《Ecology and evolution》2020,10(16):8643-8651
The more demanding requirements of DNA preservation for genomic research can be difficult to meet when field conditions limit the methodological approaches that can be used or cause samples to be stored in suboptimal conditions. Such limitations may increase rates of DNA degradation, potentially rendering samples unusable for applications such as genome‐wide sequencing. Nonetheless, little is known about the impact of suboptimal sampling conditions. We evaluated the performance of two widely used preservation solutions (1. DESS: 20% DMSO, 0.25 M EDTA, NaCl saturated solution, and 2. Ethanol >99.5%) under a range of storage conditions over a three‐month period (sampling at 1 day, 1 week, 2 weeks, 1 month, and 3 months) to provide practical guidelines for DNA preservation. DNA degradation was quantified as the reduction in average DNA fragment size over time (DNA fragmentation) because the size distribution of DNA segments plays a key role in generating genomic datasets. Tissues were collected from a marine teleost species, the Australasian snapper, Chrysophrys auratus. We found that the storage solution has a strong effect on DNA preservation. In DESS, DNA was only moderately degraded after three months of storage while DNA stored in ethanol showed high levels of DNA degradation already within 24 hr, making samples unsuitable for next‐generation sequencing. Here, we conclude that DESS was the most promising solution when storing samples for genomic applications. We recognize that the best preservation protocol is highly dependent on the organism, tissue type, and study design. We highly recommend performing similar experiments before beginning a study. This study highlights the importance of testing sample preservation protocols and provides both practical and economical advice to improve DNA preservation when sampling for genome‐wide applications. 相似文献
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ZHOU XiaoGuang REN LuFeng LI YunTao ZHANG Meng YU YuDe & YU Jun Key Laboratory of Genome Sciences Information Beijing Institute of Genomics Chinese Academy of Sciences Beijing China 《中国科学:生命科学英文版》2010,(1)
As one of the most powerful tools in biomedical research,DNA sequencing not only has been improving its productivity at an exponential growth rate but has also been evolving into a new layout of technological territories toward engineering and physical disciplines over the past three decades.In this technical review,we look into technical characteristics of the next-generation sequencers and provide insights into their future development and applications.We envisage that some of the emerging platforms are c... 相似文献
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Most plant species are recent or ancient polyploids (displaying at least one round of genome duplication in their history). Cultivated species (e.g. wheat, cotton, canola, sugarcane, coffee) and invasive species are often relatively recent polyploids, and frequently of hybrid origin (i.e. allopolyploids). Despite the genetic bottleneck occurring during the allopolyploid speciation process, the formation of such species from two divergent lineages leads to fixed heterozygosity decisive to their success. New phenotypes and new niche occupation are usually associated with this mode of speciation, as a result of both genomic rearrangements and gene expression changes of different magnitudes depending on the different polyploid species investigated. These gene expression changes affecting newly formed polyploid species may result from various, interconnected mechanisms, including (i) functional interactions between the homoeologous copies and between their products, that are reunited in the same nucleus and cell; (ii) the fate of duplicated copies, selective pressure on one of the parental copy being released which could lead to gene loss, pseudogenization, or alternatively, to subfunctionalization or neofunctionalization; and (iii) epigenetic landscape changes that in turn affect gene expression. As one of the interrelated processes leading to epigenetic regulation of gene expression, the DNA methylation status of newly formed species appears to be consistently affected following both hybridization and genome doubling. In this issue, Verhoeven et al. have investigated the fate of DNA methylation patterns that could affect naturally occurring new asexual triploid lineages of dandelions. As a result of such a ploidy level change, the authors demonstrate stably transmitted DNA methylation changes leading to unique DNA methylation patterns in each newly formed lineage. Most studies published to date on plant DNA methylation polymorphism were performed using restriction enzymes sensitive to methylation. Recently, new high‐throughput methods were made available, thanks to the development of ‘next‐generation sequencing’ techniques. The combination of these methods offers powerful and promising tools to investigate epigenetic variation in both model and non‐model systems. 相似文献
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Bisulfite sequencing (BS-seq) technology measures DNA methylation at single nucleotide resolution. A key task in BS-seq data analysis is to identify differentially methylation (DM) under different conditions. Here we provide a tutorial for BS-seq DM analysis using Bioconductor package DSS. DSS uses a beta-binomial model to characterize the sequence counts from BS-seq, and implements rigorous statistical method for hypothesis testing. It provides flexible functionalities for a variety of DM analyses. 相似文献
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Embryonic stem cells (ESCs) are pluripotent, self‐renewing cells. These cells can be used in applications such as cell therapy, drug development, disease modeling, and the study of cellular differentiation. Investigating the interplay of epigenetics, genetics, and gene expression in control of pluripotence and differentiation could give important insights on how these cells function. One of the best known epigenetic factors is DNA methylation, which is a major mechanism for regulation of gene expression. This phenomenon is mostly seen in imprinted genes and X‐chromosome inactivation where DNA methylation of promoter regions leads to repression of gene expression. Differential DNA methylation of pluripotence‐associated genes such as Nanog and Oct4/Pou5f1 has been observed between pluripotent and differentiated cells. It is clear that tight regulation of DNA methylation is necessary for normal development. As more associations between aberrant DNA methylation and disease are reported, the demand for high‐throughput approaches for DNA methylation analysis has increased. In this article, we highlight these methods and discuss recent DNA methylation studies on ESCs. J. Cell. Biochem. 109: 1–6, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
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Shin-ichiro Takebayashi Shin Ogata Masato Ogata Katsuzumi Okumura 《Bioscience, biotechnology, and biochemistry》2013,77(12):2098-2100
ABSTRACTHere, we show that semiconductor-based sequencing technology can be used to map mammalian replication domains, chromosomal units with similar DNA replication timing. Replicating DNA purified from mammalian cells was successfully sequenced by the Ion Torrent platform. The resultant replication domain map of mouse embryonic stem cells is comparable to those obtained by the conventional microarray-based method. 相似文献
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哺乳动物印记域DLK1-DIO3的研究进展 总被引:4,自引:0,他引:4
DLK1-DIO3印记域定位于人14号染色体、小鼠12号染色体及绵羊18号染色体远端, 在真哺乳亚纲动物中印记保守。该印记域包含3个编码蛋白的父系表达基因Dlk1、Rtl1和Dio3以及若干大小不同的母系表达印记非编码RNA, 如miRNAs、snoRNAs 和大型非编码RNA Gtl2等。人和小鼠该印记域内印记基因剂量的改变将导致严重的表型异常甚至胚胎致死, 暗示正常的发育需要域内印记基因的正常表达。文章重点论述了哺乳动物DLK1-DIO3印记域的印记调控机制和域内印记基因及其功能的研究进展。 相似文献
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Eric M. O'Neill Rachel Schwartz C. Thomas Bullock Joshua S. Williams H. Bradley Shaffer X. Aguilar‐Miguel Gabriela Parra‐Olea David W. Weisrock 《Molecular ecology》2013,22(1):111-129
Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome‐wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next‐generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high‐quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome‐wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi‐species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon‐based PTS to rapidly generate large‐scale data for population genetic and phylogenetic‐based research. 相似文献
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Rea M Zheng W Chen M Braud C Bhangu D Rognan TN Xiao W 《The Plant journal : for cell and molecular biology》2012,71(5):776-786
Imprinting, i.e. parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen, and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant showed that the maternal histone H1 allele is required for DME regulation of MEA, FWA and FIS2 imprinting in Arabidopsis endosperm but the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in DME-mediated DNA methylation and gene regulation at imprinted loci. 相似文献