Abstract
Efforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgonians Eunicea flexuosa and Gorgonia ventalina, and the scleractinian Porites panamensis. The 20-bp PNA clamp was designed using DNA from E. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads in E. flexuosa samples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction of E. flexuosa DNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian, G. ventalina, by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral, P. panamensis, by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities.
Highlights
Microbial diversity within a coral host can exceed thousands of species, with individual colonies hosting as many as 650 unique OTUs
Extraction protocols have been developed that increased microbial DNA yields from coral samples (Galkiewicz and Kellogg 2008; Sunagawa et al 2010; Weber et al 2017), but they still fail to completely remove host DNA
We have shown that adding peptide nucleic acid (PNA) clamps is an efficient, cost-effective way to mitigate coral host contamination leading to higher microbial read counts without introducing bias
Summary
Microbial diversity within a coral host can exceed thousands of species, with individual colonies hosting as many as 650 unique OTUs (operational taxonomic units; Bayer et al 2013; Hernandez-Agreda et al 2018). Despite the proposed importance of microorganisms in corals and the recent advances in high-throughput sequencing technology that allow for deep sequencing of bacteria and archaeal amplicons, our knowledge of the composition and function of these microbial communities remains limited. Corals cells are rife with heavy pigmentation and nucleases, both of which are PCR inhibitors (ten Lohuis et al 1990; Price and Linge 1999) These issues are problematic for coral microbiome studies because coral DNA is amplified by the bacterial specific primers employed by large-scale microbial sequencing projects (e.g., Earth Microbiome Project; Galkiewicz and Kellogg 2008; Weber et al 2017)
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