title: Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure.
abstract: PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.
authors.affiliation: Josephine Bay Paul Center, Marine Biological Laboratory, Woods Hole, MA 02543, USA. jhuber@mbl.edu
authors.forename: Julie A
authors.initials: JA
authors.lastname: Huber
authors.forename: Hilary G
authors.initials: HG
authors.lastname: Morrison
authors.forename: Susan M
authors.initials: SM
authors.lastname: Huse
authors.forename: Phillip R
authors.initials: PR
authors.lastname: Neal
authors.forename: Mitchell L
authors.initials: ML
authors.lastname: Sogin
authors.forename: David B
authors.initials: DB
authors.lastname: Mark Welch
mesh_terms: D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification*; D044822:Biodiversity; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D003951:Diagnostic Errors; D015723:Gene Library; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S