Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles.
Evaluation of the Effects of Library Preparation Procedure and Sample Characteristics on the Accuracy of Metagenomic Profiles.
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DOI：
10.1128/msystems.00440-21
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发表时间：
2021-10-26
期刊：
影响因子：
6.4
通讯作者：
Sharpton TJ
中科院分区：
文献类型：
--
作者：
Gaulke CA;Schmeltzer ER;Dasenko M;Tyler BM;Vega Thurber R;Sharpton TJ
霰弹枪宏基因组测序改变了我们对微生物群落生态学的理解。然而，制备用于高通量DNA测序的宏基因组文库仍然是昂贵的、劳动密集型的和耗时的程序，这反过来限制了宏基因组的效用。最近开发了几种图书馆准备程序来抵消这些费用，但目前还不清楚这些新程序与该领域的现行标准相比如何。特别是，尚不清楚所有这些程序是否在不同类型的微生物群落中表现同样好，或者正在处理的生物样品的特征（例如，DNA量）影响方法的准确性。为了解决这些问题，我们评估了五种不同的鸟枪DNA序列文库制备方法，包括常用的Nextera Flex试剂盒，在应用于宏基因组DNA时的表现。我们测量了每种方法产生宏基因组数据的能力，这些数据准确地代表了社区的潜在分类和遗传多样性。我们在一系列微生物群落类型（例如，土壤、珊瑚相关和小鼠肠道相关）和输入DNA量。我们发现，社区的类型和输入DNA的数量影响每个方法的性能，这表明在选择方法时可能需要仔细考虑，特别是对于低复杂度的社区。然而，我们评估的具有成本效益的制备方法通常与目前用于高复杂性社区的金标准Nextera DNA Flex试剂盒相当。总的来说，这项分析的结果将有助于扩大甚至促进未来研究中的宏基因组方法。宏基因组文库制备方法和测序技术继续快速发展，使研究人员能够表征以前未充分探索的环境样品和系统中的微生物群落。然而，广泛接受的标准化文库制备方法可能成本过高。新的可用方法可能更便宜，但与标准方法相比，它们的有效性仍然未知。在这项研究中，我们比较了五种不同的宏基因组文库制备方法。我们在一系列复杂性和输入DNA数量不同的微生物群落中评估了每种方法。我们的研究结果表明，在选择最合适的宏基因组文库制备方法时，考虑样品特性（包括群落类型、组成和DNA量）的重要性。
Shotgun metagenomic sequencing has transformed our understanding of microbial community ecology. However, preparing metagenomic libraries for high-throughput DNA sequencing remains a costly, labor-intensive, and time-consuming procedure, which in turn limits the utility of metagenomes. Several library preparation procedures have recently been developed to offset these costs, but it is unclear how these newer procedures compare to current standards in the field. In particular, it is not clear if all such procedures perform equally well across different types of microbial communities or if features of the biological samples being processed (e.g., DNA amount) impact the accuracy of the approach. To address these questions, we assessed how five different shotgun DNA sequence library preparation methods, including the commonly used Nextera Flex kit, perform when applied to metagenomic DNA. We measured each method’s ability to produce metagenomic data that accurately represent the underlying taxonomic and genetic diversity of the community. We performed these analyses across a range of microbial community types (e.g., soil, coral associated, and mouse gut associated) and input DNA amounts. We find that the type of community and amount of input DNA influence each method’s performance, indicating that careful consideration may be needed when selecting between methods, especially for low-complexity communities. However, the cost-effective preparation methods that we assessed are generally comparable to the current gold-standard Nextera DNA Flex kit for high-complexity communities. Overall, the results from this analysis will help expand and even facilitate access to metagenomic approaches in future studies. IMPORTANCE Metagenomic library preparation methods and sequencing technologies continue to advance rapidly, allowing researchers to characterize microbial communities in previously underexplored environmental samples and systems. However, widely accepted standardized library preparation methods can be cost-prohibitive. Newly available approaches may be less expensive, but their efficacy in comparison to standardized methods remains unknown. In this study, we compared five different metagenomic library preparation methods. We evaluated each method across a range of microbial communities varying in complexity and quantity of input DNA. Our findings demonstrate the importance of considering sample properties, including community type, composition, and DNA amount, when choosing the most appropriate metagenomic library preparation method.