Evaluating bias for Illumina-based bacterial 16S rRNA gene profiles

Massively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/inter-lane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soils and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5-10 ng) and low (0.1 ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability. Pooling of multiple PCR amplicons influenced the separation of all profiles from each sample and reduced within-sample heterogeneity, although these effects were not always significant. By comparison, sample preparation and inter-lane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing.

16S核糖体RNA（16S rRNA）基因的大规模平行测序，可在充足测序深度下实现陆地、水生及宿主关联微生物群落的比较分析，从而可靠评估α多样性与β多样性。建立微生物群落分析的标准化实验方案，需通过最小化方法学偏倚来源，提升基于聚合酶链式反应（PCR）的分子检测的可重复性。本研究中，我们测试了模板浓度、PCR扩增产物混样、样本制备/泳道间测序，对细菌16S rRNA基因双端Illumina测序相关可重复性的影响。我们以土壤和粪便样本的DNA提取物作为模板，分别对高（5-10 ng）与低（0.1 ng）模板浓度下的混样扩增产物与单反应扩增产物进行测序。此外，所有实验操作均在两个独立日期重复，并在两个不同的MiSeq测序泳道上完成测序。尽管样本内的序列谱高度一致，但模板浓度对样本谱的变异性具有显著影响。多个PCR扩增产物的混样操作，会改变各样本的谱间分离程度并降低样本内异质性，尽管这类效应并非总是显著。相比之下，样本制备与泳道间变异性未对样本序列数据产生显著影响。本系统性分析凸显了优化模板浓度以最小化微生物群落检测中变异性的重要性，并表明在下一代测序前对多个PCR扩增产物进行混样的操作，在降低16S rRNA基因测序检测偏倚方面的贡献相对有限。