title: Normalization of environmental metagenomic DNA enhances the discovery of under-represented microbial community members.
abstract: Normalization is a procedure classically employed to detect rare sequences in cellular expression profiles (i.e. cDNA libraries). Here, we present a normalization protocol involving the direct treatment of extracted environmental metagenomic DNA with S1 nuclease, referred to as normalization of metagenomic DNA: NmDNA. We demonstrate that NmDNA, prior to post hoc PCR-based experiments (16S rRNA gene T-RFLP fingerprinting and clone library), increased the diversity of sequences retrieved from environmental microbial communities by detection of rarer sequences. This approach could be used to enhance the resolution of detection of ecologically relevant rare members in environmental microbial assemblages and therefore is promising in enabling a better understanding of ecosystem functioning.
  
  
  CONCLUSIONS
  This study is the first testing 'normalization' on environmental metagenomic DNA (mDNA). The aim of this procedure was to improve the identification of rare phylotypes in environmental communities. Using hypoliths as model systems, we present evidence that this post-mDNA extraction molecular procedure substantially enhances the detection of less common phylotypes and could even lead to the discovery of novel microbial genotypes within a given environment.
authors.affiliation: Centre for Microbial Ecology and Genomics (CMEG), Genomic Research Institute, University of Pretoria, Pretoria, South Africa; Institute for Microbial Biotechnology and Metagenomics (IMBM), University of the Western Cape, Bellville, South Africa.
authors.forename: J-B
authors.initials: JB
authors.lastname: Ramond
authors.forename: T P
authors.initials: TP
authors.lastname: Makhalanyane
authors.forename: M I
authors.initials: MI
authors.lastname: Tuffin
authors.forename: D A
authors.initials: DA
authors.lastname: Cowan
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D017753:Ecosystem; D015723:Gene Library; D056186:Metagenomics / Q000379:methods*; D059013:Microbial Consortia / Q000235:genetics*; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: hypolith; metagenomic DNA; microbial assemblage; normalization; rare biosphere