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

Liyou Wu, Chongqing Wen, Yujia Qin, Huaqun Yin, Qichao Tu, Joy D. Van Nostrand, Tong Yuan, Menting Yuan, Ye Deng, Jizhong Zhou

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. 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. 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.