Error rate of 454 GS FLX sequenced V4 and V9 PCR amplicons of various protists

Initial environmental pyrosequencing studies suggested highly complex protistan communities with phylotype richness decisively higher than previously estimated. However, recent studies on individual bacteria or artificial bacterial communities evidenced that pyrosequencing errors may skew our view of the true complexity of microbial communities. We pyrosequenced two diversity markers (hypervariable regions V4 and V9 of the small subunit rDNA) of an intertidal protistan model community, using the Roche GS-FLX and the most recent GS-FLX Titanium sequencing systems. After pyrosequencing 24 reference sequences we obtained up to 2,039 unique tags (from 3,879 V4 GS-FLX Titanium reads), 77% of which were singletons. Even binning sequences that share 97% similarity still emulated a pseudodiversity exceeding the true complexity of the model community up to three times (V9 GS-FLX). Pyrosequencing error rates were higher for V4 fragments compared to the V9 domain and for the GS-FLX Titanium compared to the GS-FLX system. Furthermore, this experiment revealed that error rates are taxon-specific. As an outcome of this study we suggest a fast and efficient strategy to discriminate pyrosequencing signals from noise in order to more realistically depict the structure of protistan communities using simple tools that are implemented in standard tag data processing pipelines.

早期环境焦磷酸测序（pyrosequencing）研究显示，原生生物群落具有极高的复杂性，其系统型（phylotype）丰富度显著高于此前的预估。然而，近期针对单一细菌或人工构建细菌群落的研究表明，焦磷酸测序误差可能会扭曲我们对微生物群落真实复杂性的认知。本研究针对一个潮间带原生生物模式群落，利用罗氏（Roche）GS-FLX与最新的GS-FLX Titanium测序平台，对两个多样性标记基因——即小亚基rDNA（small subunit rDNA）的高变区V4与V9——进行了焦磷酸测序。在对24条参考序列进行焦磷酸测序后，我们从3879条V4 GS-FLX Titanium读段中获得了多达2039个独特标签（unique tags），其中77%为单序列（singletons）。即便将相似度达97%的序列进行聚类（binning），仍会得到超出模式群落真实复杂性达三倍之多的伪多样性（pseudodiversity）（以V9 GS-FLX数据为例）。焦磷酸测序的误差率在V4片段中高于V9区域，且在GS-FLX Titanium平台中高于GS-FLX平台。此外，本实验还发现误差率具有类群特异性（taxon-specific）。基于本研究结果，我们提出了一种快速高效的策略，可通过标准标签数据处理流程中集成的简易工具，区分焦磷酸测序的信号与噪声，从而更真实地还原原生生物群落的结构。