title: Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform.
abstract: BACKGROUND
  16S rRNA gene pyrosequencing approach has revolutionized studies in microbial ecology. While primer selection and short read length can affect the resulting microbial community profile, little is known about the influence of pyrosequencing methods on the sequencing throughput and the outcome of microbial community analyses. The aim of this study is to compare differences in output, ease, and cost among three different amplicon pyrosequencing methods for the Roche/454 Titanium platform
  
  
  RESULTS
  The following three pyrosequencing methods for 16S rRNA genes were selected in this study: Method-1 (standard method) is the recommended method for bi-directional sequencing using the LIB-A kit; Method-2 is a new option designed in this study for unidirectional sequencing with the LIB-A kit; and Method-3 uses the LIB-L kit for unidirectional sequencing. In our comparison among these three methods using 10 different environmental samples, Method-2 and Method-3 produced 1.5-1.6 times more useable reads than the standard method (Method-1), after quality-based trimming, and did not compromise the outcome of microbial community analyses. Specifically, Method-3 is the most cost-effective unidirectional amplicon sequencing method as it provided the most reads and required the least effort in consumables management.
  
  
  CONCLUSIONS
  Our findings clearly demonstrated that alternative pyrosequencing methods for 16S rRNA genes could drastically affect sequencing output (e.g. number of reads before and after trimming) but have little effect on the outcomes of microbial community analysis. This finding is important for both researchers and sequencing facilities utilizing 16S rRNA gene pyrosequencing for microbial ecological studies.
authors.affiliation: Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Tamaki
authors.forename: Chris L
authors.initials: CL
authors.lastname: Wright
authors.forename: Xiangzhen
authors.initials: X
authors.lastname: Li
authors.forename: Qiaoyan
authors.initials: Q
authors.lastname: Lin
authors.forename: Chiachi
authors.initials: C
authors.lastname: Hwang
authors.forename: Shiping
authors.initials: S
authors.lastname: Wang
authors.forename: Jyothi
authors.initials: J
authors.lastname: Thimmapuram
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Kamagata
authors.forename: Wen-Tso
authors.initials: WT
authors.lastname: Liu
mesh_terms: D004783:Environmental Microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017421:Sequence Analysis / Q000295:instrumentation* / Q000379:methods*
chemical_list: D012336:RNA, Ribosomal, 16S