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Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps
Coral Reefs ( IF 3.5 ) Pub Date : 2020-10-06 , DOI: 10.1007/s00338-020-02006-5
Alicia M. Reigel , Sarah M. Owens , Michael E. Hellberg

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.

中文翻译:

使用 PNA 夹减少珊瑚虫微生物组 16S rRNA 基因调查中的宿主 DNA 污染

由于通用细菌 16S rRNA 基因引物的宿主扩增水平高,测序过程效率低下,因此研究与珊瑚相关的微生物群落的努力可能会受到限制。在这里,我们开发了一种廉价的肽核酸 (PNA) 夹子,它在 PCR 过程中与宿主 DNA 的目标序列结合并阻止扩增。然后,我们测试了这种 PNA 夹钳减轻宿主污染和增加来自三种珊瑚物种样本的整体微生物序列覆盖率的能力:柳珊瑚 Eunicea flexuosa 和 Gorgonia ventalina,以及石珊瑚 Porites panamensis。20-bp PNA 夹是使用来自 E. flexuosa 的 DNA 设计的。在 PCR 过程中添加 PNA 夹将 E. flexuosa 样品中微生物读数的百分比增加了 11 倍以上。没有和有 PNA 夹钳的微生物群落多样性相似,十个最丰富的 ASV(扩增子序列变体)的相对频率也是如此,这表明夹钳成功地阻止了宿主 DNA 扩增,同时按比例增加了最丰富的微生物 DNA 扩增分类群。E. flexuosa DNA 的减少与稀有分类群丰度的增加相关。该钳夹还将另一种柳珊瑚 G. ventalina 中微生物读数的平均百分比增加了 8.6 倍,而不会改变微生物群落的β多样性,而在远缘相关的石珊瑚 P. panamensis 中,微生物读数的平均百分比增加了近两倍。宿主污染的减少与宿主扩增子和 PNA 夹之间的核苷酸错配数量相关。PNA 夹具的成本低至 0 美元。
更新日期:2020-10-06
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