Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis

BMC Microbiology

Jizhong Zhou⁵  Ye Deng³  Menting Yuan¹  Tong Yuan¹  Joy D. Van Nostrand¹  Qichao Tu¹  Huaqun Yin²  Yujia Qin¹  Chongqing Wen⁴  Liyou Wu¹ 

[1] Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman 73019, OK, USA;Key Laboratory of Biometallurgy of the Ministry of Education, Changsha, Hunan, China;CAS Key Laboratory of Environmnetal Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China;Fisheries College, Guangdong Ocean University, Zhanjiang, Guangdong, China;Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley 94720, CA, USA

关键词: Microbial ecology;    Phasing primer;    Microbial community;    Illumina Miseq;    Amplicon sequencing;    Low diversity sample;    Next generation sequencing;   

Others  :  1221661
DOI  :  10.1186/s12866-015-0450-4

 received in 2015-03-03, accepted in 2015-05-18,  发布年份 2015

【 摘 要 】

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

【 授权许可】

   
2015 Wu et al.

【 图 表 】

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