| Human, Bacterial and Fungal Amplicon Collection and Processing for Sequencing
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Author:
Date:
2015-05-20
[Abstract] Sequencing taxonomic marker genes is a powerful tool to interrogate the composition of microbial communities. For example, bacterial and fungal community composition can be evaluated in parallel using the 16S ribosomal RNA gene for bacteria or the internal transcribed spacer region in fungi. These are conserved regions that are universal to a taxonomic clade, yet have undergone some degree of evolution such that different lineages can be differentiated. Conserved regions are used for design of universal priming sites that allow amplification of the marker gene out of a mixed microbial community. Here, we describe our standard operating procedure to collect and sequence 16S rRNA and ITS1 amplicons from human skin. We use the 16S rRNA V1-V3 region for skin samples, as it has greater power ...
[摘要] 测序分类标记基因是一个强有力的工具，以询问微生物群落的组成。 例如，可以使用细菌的16S核糖体RNA基因或真菌中的内部转录间隔区平行评估细菌和真菌群落组成。 这些是对分类学分支是通用的保守区，但已经经历了某种程度的进化，使得不同谱系可以分化。 保守区用于设计允许从混合微生物群落扩增标记基因的通用引发位点。 在这里，我们描述我们的标准操作程序收集和序列16S rRNA和ITS1扩增子从人类皮肤。 我们使用16S rRNA V1-V3区域的皮肤样本，因为它有更大的权力分类皮肤中的常见葡萄球菌。 该方案适用于扩增子的454焦磷酸测序。
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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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