Fort Lauderdale Research and Education Center, University of Florida, IFAS, 3205 College Avenue, Fort Lauderdale, FL 33314, USA Hubbard Center for Genome Studies, University of New Hampshire, 35 Colovos Rd., Durham, NH 03824, USA.
Mol Ecol Resour. 2010 Jul;10(4):666-76. doi: 10.1111/j.1755-0998.2009.02819.x. Epub 2009 Dec 15.
Although nematodes are the most abundant metazoan animals on Earth, their diversity is largely unknown. To overcome limitations of traditional approaches (labour, time, and cost) for assessing biodiversity of nematode species in environmental samples, we have previously examined the suitability of high-throughput sequencing for assessing species level diversity with a set of control experiments employing a mixture of nematodes of known number and with known sequences for target diagnostic loci. Those initial experiments clearly demonstrated the suitability of the approach for identification of nematode taxa but lacked the replicate experiments necessary to evaluate reproducibility of the approach. Here, we analyze reads generated from three different PCR amplifications and three different sequencing reactions to examine the differential PCR amplification, the possibility of emulsion PCR artefacts, and differences between sequencing reactions. Our results suggest that both qualitative and quantitative data are consistent and highly reproducible. Variation associated with in-house PCR amplification or emPCR and sequencing are present but the representation of each nematode is very consistent from experiment to experiment and supports the use of read counts to estimate relative abundance of taxa in a metagenetic sample.
尽管线虫是地球上最丰富的后生动物，但它们的多样性在很大程度上是未知的。为了克服传统方法（劳动力、时间和成本）在评估环境样本中线虫物种生物多样性方面的局限性，我们之前已经检查了高通量测序在评估具有已知数量的线虫混合物和目标诊断基因座的已知序列的线虫物种的物种水平多样性方面的适用性。这些初步实验清楚地表明了该方法用于鉴定线虫分类群的适用性，但缺乏必要的重复实验来评估该方法的可重复性。在这里，我们分析了来自三个不同 PCR 扩增和三个不同测序反应的读取，以检查差异 PCR 扩增、乳液 PCR 假象的可能性以及测序反应之间的差异。我们的结果表明，定性和定量数据都是一致且高度可重复的。与内部 PCR 扩增或 emPCR 和测序相关的变异是存在的，但每个线虫的代表从一个实验到另一个实验非常一致，支持使用读取计数来估计宏基因组样本中分类群的相对丰度。
