P17-07 : Droplet-based 16S rRNA gene-targeted PCR improves the quantitative performance of microbial community structure determination using next-generation DNA sequencing
Massively parallel sequencing of 16S rRNA (16S) gene amplicons has become a cornerstone technique for determining the composition and structure of complex microbial communities in natural and engineered ecosystems. However, biases introduced during the multi-template 16S PCR step currently limit the accuracy of the measurement process. In this study, we evaluated the effectiveness of single-template 16S PCR in emulsion droplets to reduce amplification bias and allow more accurate measurement of microbial community structure. Using defined bacterial mock communities and various commonly used primer sets, we demonstrate that droplet-based 16S PCR can improve the agreement between expected and measured species abundances. We further established that the benefit of droplet PCR can be enhanced by increasing the number of amplification cycles, which had only a marginal effect on base-call errors. As droplet-generating systems are becoming more readily accessible, we anticipate that this approach can be a valuable tool in studies that demand accurate microbial community structure measurements. Furthermore, by inclusion of artificial 16S rRNA gene spike-ins currently being developed by our group, droplet-based 16S PCR may also enable accurate and community-wide absolute quantification using next-generation sequencing.
keywords:Microbial community analysis,PCR bias,Droplet PCR,Next-generation sequencing,Accuracy