Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer
Evaluation of PCR conditions for characterizing bacterial communities with full-length 16S rRNA genes using a portable nanopore sequencer
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DOI：
10.1038/s41598-020-69450-9
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发表时间：
2020-07-28
影响因子：
4.6
通讯作者：
Maruyama, Fumito
中科院分区：
文献类型：
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作者：
Fujiyoshi, So;Muto-Fujita, Ai;Maruyama, Fumito
MinION（Oxford Nanopore Technologies）是一种便携式纳米孔测序仪，于2014年作为一种新的DNA测序技术推出。MinION现在被广泛使用，因为它的初始启动成本相对于现有的DNA测序仪较低，具有良好的便携性，易于处理，实时分析和长读取输出。然而，基于16 S rRNA的PCR所用实验条件的差异可能会导致样本中细菌群落评估的偏差。因此，需要关于可靠实验条件的基本知识，以确保适当使用该技术。我们的研究涉及的技术的可靠性，获得准确和定量的全长16S rRNA扩增子测序数据的细菌群落结构评估使用MinION。我们使用三个独立的模拟微生物群落标准DNA比较了五种PCR条件，并在试验中建立了适当的、标准化的、更好的PCR条件。然后，我们使用Illumina MiSeq对两个模拟社区和六个环境样本进行测序以进行比较。优化后的PCR条件为95 ℃ 1 min、60 ℃ 1 min、68 ℃ 3 min，共35个循环。
MinION (Oxford Nanopore Technologies), a portable nanopore sequencer, was introduced in 2014 as a new DNA sequencing technology. MinION is now widely used because of its low initial start-up costs relative to existing DNA sequencers, good portability, easy-handling, real-time analysis and long-read output. However, differences in the experimental conditions used for 16S rRNA-based PCR can bias bacterial community assessments in samples. Therefore, basic knowledge about reliable experimental conditions is needed to ensure the appropriate use of this technology. Our study concerns the reliability of techniques for obtaining accurate and quantitative full-length 16S rRNA amplicon sequencing data for bacterial community structure assessment using MinION. We compared five PCR conditions using three independent mock microbial community standard DNAs and established appropriate, standardized, better PCR conditions among the trials. We then sequenced two mock communities and six environmental samples using Illumina MiSeq for comparison. Modifying the PCR conditions improved the sequencing quality; the optimized conditions were 35 cycles of 95 degrees C for 1 min, 60 degrees C for 1 min and 68 degrees C for 3 min. Our results provide important information for researchers to determine bacterial community using MinION accurately.