Document Type

Article

Publication Date

10-2020

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 gorgoniansEunicea flexuosaandGorgonia ventalina,and the scleractinianPorites panamensis. The 20-bp PNA clamp was designed using DNA fromE. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads inE. flexuosasamples 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 ofE. flexuosaDNA 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. Keywords

Publication Source (Journal or Book title)

CORAL REEFS

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