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| Influence of DNA Extraction Method on Microbial Diversity Analysis Through Next Generation Sequencing |
| AN Yun-he1,2, CHENG Xiao-yan1,2, TIAN Yan-jie1,2, MA Kai1,2, GAO Li-juan1,2, WU Hui-juan1,2 |
1. Beijing Center For Physical and Chemical Analysis, Beijing 100089, China;
2. Beijing Engineering Technique Research Center for Gene Sequencing & Function Analysis, Beijing 100094, China |
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Abstract Using of high throughput sequencing technology to study the microbial diversity in complex samples has become one of the most hot issues in the field of microbial research. In this new technology, the microbial sample preparation, such as DNA extraction and the amplification of 16S variable region, etc., is crucial for the data analysis, especially for the microbial community composition analysis. The soil and sheep rumen chyme samples were used to extract DNA by domestic Kit (TIANamp soil DNA Kit) and imported kit (PowerSoil DNA isolation kit) respectively. Then the 25ng total DNA was used to amplify the 16S V3 region, and the final sequencing library was constructed by mixing equal amounts of purified PCR products. Finally, the OTU amount,rarefaction curve, microbial number and species were compared by data analysis. It was found that under the same amount of DNA template and PCR cycle number, the DNA treated by imported kit can obtain more microbial species. But under the same DNA extraction kit and DNA template amount, the proportion of the community composition was not the best with more PCR cycle numbers, although the number of species was much more. This may be due to the excessive expansion of the inferior bacteria, which lead to the microbial composition imbalance.
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Received: 20 September 2017
Published: 15 November 2017
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