| Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
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Author:
Date:
2018-08-05
[Abstract] Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was ...
[摘要] 染色体构象捕获测序（Hi-C）是一种全面询问细胞核中染色质三维定位的有效方法。 Hi-C的发展可以追溯到染色体构象捕获的分辨率和通量的连续增加（3C）（Dekker et al。，2002）。 3C的基本工作流程包括（i）通常用甲醛固定完整的染色质，（ii）用限制酶切割固定的染色质，（iii）在稀释条件下重新连接粘性末端，以促进交联片段之间的连接或随机片段之间的那些和（iv）量化基因组基因座对之间的连接事件的数量（de Wit和de Laat，2012）。在最初的3C方案中，通过半定量PCR扩增对应于少量基因组位点（“一对一”）的选定连接接头来测量连接频率（Dekker et al。，2002 ）。然后，染色体构象捕获芯片（4C）和染色体构象捕获碳复制（5C）技术扩展3C以分别以“一对多”或“多对多”方式计算结扎事件。 Hi-C（Lieberman-Aiden et al。，2009）最终将3C与下一代测序相结合（Metzker，2010）。此处，在再连接之前，用生物素标记的核苷酸类似物填充粘性末端以在后续步骤中富集具有连接连接的片段。然后对Hi-C文库进行高通量测序，并将得到的读数映射到参考基因组，允许以“多对多”方式确定接触概率，其分辨率仅受限制性位点的分布限制和阅读深度。 Hi-C的首次应用是阐明人类基因组中的全球染色质折叠原理（Lieberman-Aiden et ...
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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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