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Wu L.,University of Oklahoma | Wen C.,Guangdong Ocean University | Qin Y.,University of Oklahoma | Yin H.,Central South University | And 7 more authors.
BMC Microbiology

Background: Although high-throughput sequencing, such as Illumina-based technologies (e.g. MiSeq), has revolutionized microbial ecology, adaptation of amplicon sequencing for environmental microbial community analysis is challenging due to the problem of low base diversity. Results: A new phasing amplicon sequencing approach (PAS) was developed by shifting sequencing phases among different community samples from both directions via adding various numbers of bases (0-7) as spacers to both forward and reverse primers. Our results first indicated that the PAS method substantially ameliorated the problem of unbalanced base composition. Second, the PAS method substantially improved the sequence read base quality (an average of 10 % higher of bases above Q30). Third, the PAS method effectively increased raw sequence throughput (~15 % more raw reads). In addition, the PAS method significantly increased effective reads (9-47 %) and the effective read sequence length (16-96 more bases) after quality trim at Q30 with window 5. In addition, the PAS method reduced half of the sequencing errors (0.54-1.1 % less). Finally, two-step PCR amplification of the PAS method effectively ameliorated the amplification biases introduced by the long barcoded PCR primers. Conclusion: The developed strategy is robust for 16S rRNA gene amplicon sequencing. In addition, a similar strategy could also be used for sequencing other genes important to ecosystem functional processes © 2015 Wu et al. Source

Hu Q.,Central South University | Hu Q.,Key Laboratory of Biometallurgy of the Ministry of Education | Guo X.,Central South University | Guo X.,Key Laboratory of Biometallurgy of the Ministry of Education | And 10 more authors.
Research in Microbiology

The microbial community in a biological heap leaching (BHL) system is crucial for the decomposition of ores. However, the microbial community structure and functional differentiation in different parts of a biological heap leaching system are still unknown. In this study, metagenomic sequencing was used to fully illuminate the microbial community differentiation in the pregnant leach solution (PLS) and leaching heap (LH) of a BHL system. Long-read sequences (1.3 million) were obtained for the two samples, and the MG_RAST server was used to perform further analysis. The taxa analysis results indicated that the dominant genera of PLS is autotrophic bacterium Acidithiobacillus, but heterotrophic bacterium Acidiphilium is predominant in LH. Furthermore, functional annotation and hierarchical comparison with different reference samples showed that the abundant presence of genes was involved in transposition, DNA repair and heavy metal transport. The sequences related to transposase, which is important for the survival of the organism in the hostile environment, were both mainly classified into Acidiphilium for PLS and LH. These results indicated that not only autotrophic bacteria such as Acidithiobacillus, but also heterotrophic bacteria such as Acidiphilium, were essential participants in the bioleaching process. This new meta-view research will further facilitate the effective application of bioleaching. © 2015 Institut Pasteur. Source

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