Size Does Matter: Application-driven Approaches for Soil Metagenomics.
Size Does Matter: Application-driven Approaches for Soil Metagenomics.
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DOI：
10.1016/j.soilbio.2010.07.021
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发表时间：
2010-11-01
影响因子：
9.7
通讯作者：
Liles, Mark R.
中科院分区：
文献类型：
--
作者：
Kakirde, Kavita S.;Parsley, Larissa C.;Liles, Mark R.
关键词：
宏基因组分析可以提供广泛的信息的结构，组成和预测的基因功能的不同环境微生物组合。每种环境都对宏基因组研究提出了自己独特的挑战，需要专门设计的方法来适应每种环境特有的物理化学和生物因素，这些因素可能会造成技术障碍和/或使宏基因组分析产生偏差。特别是，土壤中蕴藏着异常多样的原核生物，这些原核生物在核糖体型水平之外基本上未被描述，并且是天然产物发现的潜在巨大资源。土壤宏基因组学方法的成功应用取决于选择合适的DNA提取、纯化以及必要时克隆方法用于预期的下游分析。宏基因组研究中最重要的技术考虑因素包括获得足够产量的代表环境样品或富集内的靶微生物的高纯度DNA，以及（如果需要）在合适的载体和宿主中构建宏基因组文库。在克隆文库内的平均插入大小或高通量测序方法的序列读段长度的背景下，大小确实很重要。选择适当的宏基因组筛选策略来解决感兴趣的特定问题也是必要的，这应该推动宏基因组项目早期阶段使用的方法的选择（例如，DNA大小，克隆或不克隆）。在这里，我们提出了土壤宏基因组学的前景和问题的性质，并讨论了在选择土壤采样，DNA提取，纯化和克隆方法时应考虑的因素，以实现基于最终的研究目标。
Metagenomic analyses can provide extensive information on the structure, composition, and predicted gene functions of diverse environmental microbial assemblages. Each environment presents its own unique challenges to metagenomic investigation and requires a specifically designed approach to accommodate physicochemical and biotic factors unique to each environment that can pose technical hurdles and/or bias the metagenomic analyses. In particular, soils harbor an exceptional diversity of prokaryotes that are largely undescribed beyond the level of ribotype and are a potentially vast resource for natural product discovery. The successful application of a soil metagenomic approach depends on selecting the appropriate DNA extraction, purification, and if necessary, cloning methods for the intended downstream analyses. The most important technical considerations in a metagenomic study include obtaining a sufficient yield of high-purity DNA representing the targeted microorganisms within an environmental sample or enrichment and (if required) constructing a metagenomic library in a suitable vector and host. Size does matter in the context of the average insert size within a clone library or the sequence read length for a high-throughput sequencing approach. It is also imperative to select the appropriate metagenomic screening strategy to address the specific question(s) of interest, which should drive the selection of methods used in the earlier stages of a metagenomic project (e.g., DNA size, to clone or not to clone). Here, we present both the promising and problematic nature of soil metagenomics and discuss the factors that should be considered when selecting soil sampling, DNA extraction, purification, and cloning methods to implement based on the ultimate study objectives.
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作者：
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DOI：
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发表时间：
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