A low-cost pipeline for soil microbiome profiling.
A low-cost pipeline for soil microbiome profiling.
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DOI：
10.1002/mbo3.1133
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发表时间：
2020-12
期刊：
影响因子：
3.4
通讯作者：
Clark MD
中科院分区：
文献类型：
--
作者：
Bollmann-Giolai A;Giolai M;Heavens D;Macaulay I;Malone J;Clark MD
环境和作物微生物组研究的常见瓶颈是基因组 DNA 提取和测序文库构建所需的耗材和人员成本。这对于富含聚合酶链反应 (PCR) 抑制剂的土壤等具有挑战性的环境样品来说更加困难。为了解决这个问题，我们建立了一种低成本的土壤样本基因组 DNA 提取方法。我们还提出了使用常见实验室设备的 Illumina 兼容的 16S 和 ITS rRNA 基因扩增子文库制备工作流程。我们针对两种领先的商业土壤基因组 DNA 试剂盒（MoBio PowerSoil® 和 MP Biomedicals™ FastDNA™ SPIN）以及 Zou 等人最近发布的非商业提取方法评估了我们的基因组 DNA 提取方法的性能。 （公共科学图书馆生物学，15，e2003916，2017）。我们的基准测试使用四种不同的土壤类型（针叶林、阔叶林和混交林加上标准化的谷类作物堆肥混合物），通过分析 16S V4 和 ITS rRNA 扩增子的序列变异来评估提取的基因组 DNA 的质量和数量。我们发现我们的基因组 DNA 提取方法在 DNA 质量和数量方面与市售基因组 DNA 提取试剂盒相当。 MoBio PowerSoil® 试剂盒依靠基于硅胶柱的 DNA 提取和大量清洗，可提供最干净的基因组 DNA，例如最佳的 A260:A280 和 A260:A230 吸光度比。 MP Biomedicals™ FastDNA™ SPIN 试剂盒使用大量结合材料，产生了最多的基因组 DNA。我们的方法介于两种商业试剂盒之间，可产生良好的产量和干净的基因组 DNA，片段大小约为 10 kb。对检测到的扩增子序列变异的比较分析表明，我们的方法与两种商业试剂盒具有良好的相关性。在这里，我们提出了一种用于土壤样品的低成本基因组 DNA 提取方法，该方法可以与 Illumina 兼容的简单两步扩增子文库构建工作流程结合使用，用于 16S V4 和 ITS 标记基因。我们的方法以商业试剂盒成本的一小部分提供高质量的基因组 DNA，并实现具有成本效益的大规模扩增子测序项目。值得注意的是，我们提取的 gDNA 分子足够长，适合下游技术，例如全基因测序，甚至使用长读长（PacBio 或 Nanopore）、10x Genomics 链接读长和 Dovetail 基因组学的宏基因组鸟枪方法。我们提出了一种廉价且高通量的基于磁性颗粒的土壤基因组 DNA (gDNA) 提取方法“SDE”（0.36 美元），用于下一代测序质量的 gDNA 提取，可扩展至数千个样本，只需很少的动手时间。 SDE 还可以与我们的低成本扩增子文库构建工作流程（16S，ITS，2.5 美元）结合使用，一次最多可以复用 2304 个文库。将 SDE 和我们的定制文库构建工作流程相结合，可以利用最先进的 Illumina 测序平台（例如 NovaSeq）进行大规模微生物组研究。
Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low‐cost genomic DNA extraction method for soil samples. We also present an Illumina‐compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non‐commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad‐leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column‐based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low‐cost genomic DNA extraction method for soil samples that can be coupled to an Illumina‐compatible simple two‐step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high‐quality genomic DNA at a fraction of the cost of commercial kits and enables cost‐effective, large‐scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics. We present an inexpensive and high‐throughput magnetic particle‐based soil genomic DNA (gDNA) extraction method "SDE" ($0.36) for next‐generation sequencing quality gDNA extraction, scalable to thousands of samples with little hands‐on time. SDE can also be used in combination with our low‐cost amplicon library construction workflow (16S, ITS, $2.5), enabling multiplexing of up to 2304 libraries at a time. Combining SDE and our custom library construction workflow allows utilization of state‐of‐the‐art Illumina sequencing platforms (e.g., NovaSeq) for large‐scale microbiome studies.
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Nusbaum C