Improved microbiome profiling using emulsion PCR and long-read sequencing
A well-functioning microbiome is required for normal development and health in plants, animals and humans. Yet microbiomes with altered composition can result from or cause disease. Culture-independent, high-throughput molecular microbiome profiling methods are revealing staggering microbial diversity. Yet current methods are subject to severe limitations due to taxonomic biases, chimera formation and sequencing error. Hence, true microbial species richness and abundanc cannot be determined. The central goal of this proposal is to test a novel strategy to alleviate taxonomic biases and chimera formation. This will lead to more accurate microbial profiling and accelerate efforts to understand the role of the microbiome in human health, crop production and ecosystem function. There are two specific aims: 1. Identify methods that reduce taxonomic bias in microbiome profiling. 2. Identify methods that reduce chimera formation in microbiome profiling. These biases arise from properties of PCR. For example, polymerases are more likely to initiate elongation from perfectly matching primers. This leads to taxonomic biases because primers can never perfectly match all microbial taxa. Shorter fragments are more likely to be fully copied than are longer fragments due to limited processivity, leading to biases toward taxa with short amplicons. Chimeras occur when incomplete copies compete with primers for initiation of subsequent cycles of synthesis. One theme unites these disparate phenomena: competition among templates. Droplet digital PCR (ddPCR) is a recent method whereby a single PCR is divided into miniature reactions that proceed independently. ddPCR is an alternative to qPCR for gene quantification but has not been applied to microbiome work. In ddPCR, template concentration is lowered such that a fraction of droplets lack a template and positive versus negative droplets can be counted. The innovation of the approach proposed here is that the subdivision of the reaction into millions of microdroplets reduces template competition, which should improve profile accuracy. First, fungal mock communities will be constructed and subject to short and long amplicons PCR. Otherwise identical PCRs will be carried out using standard versus emulsion ddPCR (BioRad QX200 system). High-throughput sequencing of the PCR products will be done on the Illumina NextSeq500. The ribosomal sequences and expected abundances are known a priori, permitting tests of the impacts of amplicon length and PCR method on profile accuracy.