Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data.
Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data.
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作者：
Palmer JM;Jusino MA;Banik MT;Lindner DL
保守DNA区域的高通量扩增子测序（HTAS）是表征微生物群落的有力技术。最近，spike-in模拟社区已被用于测量测序平台和数据分析管道的准确性。为了评估使用真菌内部转录间隔区（ITS）扩增子的测序平台和数据处理管道的能力，我们创建了两个由质粒中的克隆DNA组成的ITS掺入对照模拟社区：生物模拟社区，由来自真菌分类群的ITS序列组成，和合成模拟社区（SynMock），由非生物ITS样序列组成。使用这些加标对照，我们表明：（1）非生物合成对照（例如，SynMock）是参数化生物信息学管道的最佳解决方案，（2）可变长度扩增子的预聚类步骤至关重要，（3）偏倚的主要来源归因于初始聚合酶链式反应（PCR），因此HTAS读段丰度通常不代表起始值。我们开发了AMPtk，这是一种多功能软件解决方案，用于处理可变长度扩增子和基于加标对照的质量过滤HTAS数据。虽然我们在本文中描述了用于ITS序列的非生物SynMock社区，但该概念和AMPtk软件可以广泛应用于任何HTAS数据集以提高数据质量。
High-throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal internal transcribed spacer (ITS) amplicons, we created two ITS spike-in control mock communities composed of cloned DNA in plasmids: a biological mock community, consisting of ITS sequences from fungal taxa, and a synthetic mock community (SynMock), consisting of non-biological ITS-like sequences. Using these spike-in controls we show that: (1) a non-biological synthetic control (e.g., SynMock) is the best solution for parameterizing bioinformatics pipelines, (2) pre-clustering steps for variable length amplicons are critically important, (3) a major source of bias is attributed to the initial polymerase chain reaction (PCR) and thus HTAS read abundances are typically not representative of starting values. We developed AMPtk, a versatile software solution equipped to deal with variable length amplicons and quality filter HTAS data based on spike-in controls. While we describe herein a non-biological SynMock community for ITS sequences, the concept and AMPtk software can be widely applied to any HTAS dataset to improve data quality.
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