title: Phasing amplicon sequencing on Illumina Miseq for robust environmental microbial community analysis.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. lwu@rccc.ou.edu.
authors.forename: Liyou
authors.initials: L
authors.lastname: Wu
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. wphage@gmail.com.|Fisheries College, Guangdong Ocean University, Zhanjiang, Guangdong, China. wphage@gmail.com.
authors.forename: Chongqing
authors.initials: C
authors.lastname: Wen
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. yujia.qin-10@ou.edu.
authors.forename: Yujia
authors.initials: Y
authors.lastname: Qin
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. yinhuaqun@gmail.com.|School of Minerals Processing and Bioengineering, Central South University, Changsha, Hunan, China. yinhuaqun@gmail.com.|Key Laboratory of Biometallurgy of the Ministry of Education, Changsha, Hunan, China. yinhuaqun@gmail.com.
authors.forename: Huaqun
authors.initials: H
authors.lastname: Yin
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. qichao@ou.edu.
authors.forename: Qichao
authors.initials: Q
authors.lastname: Tu
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. joy.vannostrand@ou.edu.
authors.forename: Joy D
authors.initials: JD
authors.lastname: Van Nostrand
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. tyuan@ou.edu.
authors.forename: Tong
authors.initials: T
authors.lastname: Yuan
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. maggieyuan@ou.edu.
authors.forename: Menting
authors.initials: M
authors.lastname: Yuan
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. dengye1210@hotmail.com.|CAS Key Laboratory of Environmnetal Biotechnology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China. dengye1210@hotmail.com.
authors.forename: Ye
authors.initials: Y
authors.lastname: Deng
authors.affiliation: Institute for Environmental Genomics, and Department of Microbiology and Plant Biology, University of Oklahoma, Norman, OK, 73019, USA. jzhou@ou.edu.|State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Tsinghua University, Beijing, 100084, China. jzhou@ou.edu.|Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA. jzhou@ou.edu.
authors.forename: Jizhong
authors.initials: J
authors.lastname: Zhou
mesh_terms: D001105:Archaea / Q000145:classification* / Q000235:genetics; D001419:Bacteria / Q000145:classification* / Q000235:genetics; D017931:DNA Primers; D019641:DNA, Archaeal / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis*; D004783:Environmental Microbiology; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012463:Saliva / Q000382:microbiology; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D019641:DNA, Archaeal; D004269:DNA, Bacterial; D004275:DNA, Ribosomal