title: Reanalyze unassigned reads in Sanger based metagenomic data using conserved gene adjacency.
abstract: BACKGROUND
  Investigation of metagenomes provides greater insight into uncultured microbial communities. The improvement in sequencing technology, which yields a large amount of sequence data, has led to major breakthroughs in the field. However, at present, taxonomic binning tools for metagenomes discard 30-40% of Sanger sequencing data due to the stringency of BLAST cut-offs. In an attempt to provide a comprehensive overview of metagenomic data, we re-analyzed the discarded metagenomes by using less stringent cut-offs. Additionally, we introduced a new criterion, namely, the evolutionary conservation of adjacency between neighboring genes. To evaluate the feasibility of our approach, we re-analyzed discarded contigs and singletons from several environments with different levels of complexity. We also compared the consistency between our taxonomic binning and those reported in the original studies.
  
  
  RESULTS
  Among the discarded data, we found that 23.7 ± 3.9% of singletons and 14.1 ± 1.0% of contigs were assigned to taxa. The recovery rates for singletons were higher than those for contigs. The Pearson correlation coefficient revealed a high degree of similarity (0.94 ± 0.03 at the phylum rank and 0.80 ± 0.11 at the family rank) between the proposed taxonomic binning approach and those reported in original studies. In addition, an evaluation using simulated data demonstrated the reliability of the proposed approach.
  
  
  CONCLUSIONS
  Our findings suggest that taking account of conserved neighboring gene adjacency improves taxonomic assignment when analyzing metagenomes using Sanger sequencing. In other words, utilizing the conserved gene order as a criterion will reduce the amount of data discarded when analyzing metagenomes.
authors.affiliation: Biodiversity Research Center, Academia Sinica, Taipei, Taiwan.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Weng
authors.forename: Chien-Hao
authors.initials: CH
authors.lastname: Su
authors.forename: Ming-Tsung
authors.initials: MT
authors.lastname: Hsu
authors.forename: Tse-Yi
authors.initials: TY
authors.lastname: Wang
authors.forename: Huai-Kuang
authors.initials: HK
authors.lastname: Tsai
authors.forename: Daryi
authors.initials: D
authors.lastname: Wang
mesh_terms: D000465:Algorithms; D001483:Base Sequence; D016208:Databases, Factual; D023061:Gene Order; D056186:Metagenomics / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*