| Reduced Representation Bisulfite Sequencing in Maize
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Author:
Date:
2018-03-20
[Abstract] DNA methylation is an epigenetic modification that regulates plant development (Law and Jacobsen, 2010). Whole genome bisulfite sequencing (WGBS) is a state-of-the-art method for profiling genome-wide methylation patterns with single-base resolution (Cokus et al., 2008). However, for an organism with a large genome, e.g., the 2.1 Gb genome of maize, WGBS may be very expensive. Reduced representation bisulfite sequencing (RRBS) has been developed in mammalian studies (Smith et al., 2009). By digesting the genome with MspI with a size selection range of approximately 40-220 bp, CG-rich regions covering only ~1% of the human genome can be specifically sequenced. However, unlike mammalian genomes, plant genomes do not exhibit clear CpG islands. Therefore ...
[摘要] DNA甲基化是调节植物发育的表观遗传修饰（Law and Jacobsen，2010）。全基因组亚硫酸氢盐测序（WGBS）是用单碱基分辨率分析全基因组甲基化模式的最先进的方法（Cokus et al。，2008）。然而，对于具有大基因组的生物体，例如玉米的2.1Gb基因组，WGBS可能非常昂贵。代表性亚硫酸氢盐测序（RRBS）已经在哺乳动物研究中发展（Smith等人，2009）。通过用大小选择范围大约40-220bp的 Msp 消化基因组，可以对仅涵盖〜1％人类基因组的CG富含区域进行特异性测序。然而，与哺乳动物基因组不同，植物基因组不显示清楚的CpG岛。因此原来的RRBS协议不适用于工厂。因此，我们开发了一种计算机管道来选择特定的酶以生成感兴趣区域（ROI） - 富集的，例如，富含启动子的，减少的植物表达基因组（例如， Hsu et al。，2017）。通过用MseI消化玉米基因组并选择40-300bp片段，我们测序了大约四分之一的玉米基因组，同时保留了84.3％的启动子信息。该协议已在玉米中成功建立，可广泛应用于任何基因组。我们的计算机管道系统与RRBS文库制备方案相结合，允许进行计算分析和实验验证。
【背景】DNA甲基化是一种可遗传的表观遗传修饰，通过调节基因表达和染色质结构在动物，植物和真菌的许多发育过程中发挥重要作用（Law and ...
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| Whole Genome Bisulfite Sequencing and DNA Methylation Analysis from Plant Tissue
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Author:
Date:
2015-02-20
[Abstract] This protocol describes whole genome bisulfite-sequencing library preparation from plant tissue and subsequent data analysis. Allele-specific methylation analysis and genome-wide identification of differentially methylated regions are additional features of the analysis procedure.
[摘要] 该协议描述了植物组织的全基因组亚硫酸氢盐测序文库制备和随后的数据分析。 等位基因特异性甲基化分析和差异甲基化区域的全基因组鉴定是分析程序的附加特征。
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| Plant Sequence Capture Optimised for Illumina Sequencing
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Author:
Date:
2014-07-05
[Abstract] Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole ...
[摘要] 植物序列捕获用于复杂基因组（例如大麦及其亲属）的整个外显子（基因组的所有外显子）的靶向重测序（Mascher等人，2013）。测序和计算成本显着降低，因为只有大麦基因组的大量富集和基因编码部分被靶向，其仅对应于整个基因组的1-2％。因此，大大促进了诸如遗传多样性研究和单个基因的分离（"通过测序克隆"）的应用。这里，提供了描述来自基因组大麦DNA的Shotgun DNA文库的构建以在Illumina HiSeq/MiSeq系统上测序的方案。鸟枪DNA测序文库与包含大麦整个外显子组的寡核苷酸池（Exome Library）杂交。外显子组库作为包含生物素化探针（Roche/NimbleGen）的液体阵列提供。随后，使用链霉亲和素包被的磁珠对与Exome文库杂交的基因组鸟枪DNA片段进行亲和纯化。捕获的文库被PCR扩增和测序，使用高通量短读序列合成
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