Quality-Score Refinement of SSU rRNA Gene Pyrosequencing Differs Across Gene Region for Environmental Samples

• Published in: Microbial ecology 2012-08, Vol.64 (2), p.499-508
• Main Authors: De León, Kara Bowen, Ramsay, Bradley D., Fields, Matthew W.
• Format: Article
• Language: English
• Subjects: ambiguous nucleotide; Bacteria - classification; Bacteria - genetics; BASIC BIOLOGICAL SCIENCES; Biological and medical sciences; Biomedical and Life Sciences; clone library; Diversity indices; Ecology; Ecoregions; Ecosystem; Fundamental and applied biological sciences. Psychology; Genes, rRNA - genetics; Geoecology/Natural Processes; Hot Springs - microbiology; Libraries; Life Sciences; METHODS; Microbial Ecology; Microbiology; Nature Conservation; Nucleic acids; Nucleotides; Polymerase chain reaction; prime error; Rarefaction; RNA, Ribosomal, 16S - genetics; rRNA genes; Sequence Analysis, DNA - methods; Sequence Analysis, DNA - standards; Sequencing; Soil Microbiology; Species; Species diversity; Species richness; species richness estimate; Species Specificity; Water Quality/Water Pollution
• ISSN: 0095-3628; 1432-184X
• DOI: 10.1007/s00248-012-0043-9

Due to potential sequencing errors in pyrosequencing data, species richness and diversity indices of microbial systems can be miscalculated. The "traditional" sequence refinement method is not sufficient to account for overestimations (e.g., length, primer errors, ambiguous nucleotides). Recent in silico and single-organism studies have revealed the importance of sequence quality scores in the estimation of ecological indices; however, this is the first study to compare quality-score stringencies across four regions of the SSU rRNA gene sequence (V1V2, V3, V4, and V6) with actual environmental samples compared directly to corresponding clone libraries produced from the same primer sets. The nucleic acid sequences determined via pyrosequencing were subjected to varying quality-score cutoffs that ranged from 25 to 32, and at each quality-score cutoff, either 10 or 15 % of the nucleotides were allowed to be below the cutoff. When species richness estimates were compared for the tested samples, the cutoff values of Q27 15% , Q30 10% , and Q32 15% for VIV2, V4, and V6, respectively, estimated similar values as obtained with clone libraries and Sänger sequencing. The most stringent Q tested (Q32 10% ) was not enough to account for species richness inflation of the V3 region pyrosequence data. Results indicated that quality-score assessment greatly improved estimates of ecological indices for environmental samples (species richness and α-diversity) and that the effect of qualityscore filtering was region-dependent.