Nematode Control Metagenetic Samples
Reproducibility of Read Numbers in High-Throughput Sequencing Analysis of Nematode Community Composition and Structure
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.