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首页> 外文期刊>Microorganisms >The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure
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The Impact of Primer Design on Amplicon-Based Metagenomic Profiling Accuracy: Detailed Insights into Bifidobacterial Community Structure

机译：引物设计对基于扩增子的偏析性分析精度的影响：对双歧杆菌群落结构的详细洞察

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Next Generation Sequencing (NGS) technologies have overcome the limitations of cultivation-dependent approaches and allowed detailed study of bacterial populations that inhabit the human body. The consortium of bacteria residing in the human intestinal tract, also known as the gut microbiota, impacts several physiological processes important for preservation of the health status of the host. The most widespread microbiota profiling method is based on amplification and sequencing of a variable portion of the 16S rRNA gene as a universal taxonomic marker among members of the Bacteria domain. Despite its popularity and obvious advantages, this 16S rRNA gene-based approach comes with some important limitations. In particular, the choice of the primer pair for amplification plays a major role in defining the accuracy of the reconstructed bacterial profiles. In the current study, we performed an in silico PCR using all currently described 16S rRNA gene-targeting primer pairs (PP) in order to assess their efficiency. Our results show that V3, V4, V5, and V6 were the optimal regions on which to design 16S rRNA metagenomic primers. In detail, PP39 (Probio_Uni/Probio_Rev), PP41 (341F/534R), and PP72 (970F/1050R) were the most suitable primer pairs with an amplification efficiency of 98.5%. Furthermore, the Bifidobacterium genus was examined as a test case for accurate evaluation of intra-genus performances at subspecies level. Intriguingly, the in silico analysis revealed that primer pair PP55 (527f/1406r) was unable to amplify the targeted region of any member of this bacterial genus, while several other primer pairs seem to rather inefficiently amplify the target region of the main bifidobacterial taxa. These results highlight that selection of a 16S rRNA gene-based PP should be done with utmost care in order to avoid biases in microbiota profiling results.
机译：下一代测序（NGS）技术已经克服了培养依赖性方法的局限性，并允许对人体居住的细菌种群进行详细研究。栖息在人肠道中的细菌联盟，也称为肠道微生物群，影响若干重要的生理过程，以保护宿主的健康状况。最广泛的微生物谱分析方法基于16S rRNA基因的可变部分的扩增和测序作为细菌结构域成员之间的通用分类标志物。尽管其受欢迎程度和明显的优势，但这16S rRNA基因的方法具有一些重要的局限性。特别地，用于扩增的引物对的选择在定义重建的细菌谱的准确性方面起着重要作用。在目前的研究中，我们使用当前描述的16S rRNA基因靶向引物（PP）进行了硅PCR，以评估它们的效率。我们的结果表明，V3，V4，V5和V6是设计16S RRNA代理底漆的最佳区域。详细地，PP39（probio_uni / probio_rev），pp41（341f / 534r）和pp72（970f / 1050r）是最合适的引物对，扩增效率为> 98.5％。此外，将双歧杆菌属被检查为准确评估亚种水平的基因内表演的准确评价。有趣的是，硅分析显示，引物对PP55（527F / 1406R）不能扩增该细菌属的任何成员的靶向区域，而几种其他引物对似乎相当低地扩增了主要双歧杆菌类​​别的目标区域。这些结果突出显示了16S rRNA基因的PP的选择应尽最大谨慎地进行，以避免微生物群分析结果中的偏差。

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