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General Session 3: Biological oceanography & global change |
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Blocking PCR amplification of metazoan DNA for characterization of microbial eukaryotic diversity
GS3-53-S
Shangjin Tan* , The Hong Kong University of Science and Technology
Hongbin Liu, The Hong Kong University of Science and Technology
Presenter Email: stanac@connect.ust.hk
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| Microbial eukaryotes play fundamental ecological roles in marine ecosystems. However, molecular analyses of microbial eukaryotic diversity are sometimes limited or even biased because of the contamination by the high abundance of metazoan sequences. The usually used method, size-fractionation, misses the diversity and function of eukaryotes larger than this size. In this study, we adopted metazoa-specific blocking primers to selectively suppress the amplification of metazoan DNA for the characterization of microbial eukaryotic diversity using universal primers in PCR. First, quantitative PCR (qPCR) was used to optimize the design and performance of the blocking primers. Then, 454 pyrosequencing was applied to investigate the microbial eukaryotic diversity from a less studied size-fraction (> 10 µm) with samples collected from the Northwest Pacific Ocean. Results showed that the blocking efficiency was up to 72.4% with high fidelity. Impressively, the diversity was as high as that from the 0.2-10 µm fraction. Moreover, compared to the 0.2-10 µm fraction, some newly identified phylotypes, belonging to Amoebozoa and Rhizaria, were only found in the >10 µm fraction. The application of blocking primers is a promising tool to enhance the investigation of microbial eukaryotic diversity. |
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