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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative Ultra-High-Throughput Profiling of Fungal Communities
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Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative Ultra-High-Throughput Profiling of Fungal Communities

机译：内部转录的特定于间隔物的引物的改进选择可实现真菌社区的定量超高通量分析

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Ultra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predicted in silico and by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.
机译：较短的读取长度和引物扩增偏差限制了真菌群落的超高通量测序（HTS），从而减慢了对真菌群落谱进行更新的测序技术的采用。为了解决这些问题，我们评估了几种常见的内部转录间隔子（ITS）引物的性能，并设计了一种新颖的引物组和工作流程，用于同时进行真菌菌落的定量和物种水平研究。通过计算机模拟并通过对混合酵母物种的“模拟群落”进行测序来预测引物的比较和验证，以探索扩增子长度和扩增偏差对重建定义的酵母群落结构的挑战。该引物组的扩增子大小和分布均小于所有现有ITS引物组，从而通过超短扩增子，高通量测序平台最大化了高变ITS域的测序覆盖率。通过用这些引物将qPCR替换为标准PCR，此功能还可以将定量PCR（qPCR）可选地直接集成到HTS的准备工作流程中，从而对单个社区成员进行定量。此处描述的完整工作流程，利用任何经过评估的合格引物对，都可以快速剖析混合真菌群落，并有能力重建特征鲜明的啤酒和葡萄酒发酵真菌群落。

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