Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps
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Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps Alicia M. Reigel1
•
Sarah M. Owens2 • Michael E. Hellberg1
Received: 30 March 2020 / Accepted: 27 September 2020 Ó The Author(s) 2020
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 Topic Editor Carly Kenkel
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00338-020-02006-5) contains supplementary material, which is available to authorized users. & Alicia M. Reigel [email protected] 1
Department of Biological Sciences, Louisiana State University, 202 Life Sciences Bldg, Baton Rouge, LA 70803, USA
2
Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue, Lemont, IL 60439, USA
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. Keywords Coral Microbial communities Host–microbe symbiosis Holobiont PNA clamp 16S rRNA gene sequencing
Introduction 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). Coral- associated microbial symbionts likely play key roles in host biology, such as organic molecule cycling (Wegley et al. 2007; Kimes et al. 2010; Bourne et al. 2016) and production
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