skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Journal Article · · Microbial Ecology
 [1];  [2];  [2]
  1. Montana State Univ., Bozeman, MT (United States). Dept. of Microbiology. Center for Biofilm Engineering. Thermal Biology Inst.
  2. Montana State Univ., Bozeman, MT (United States). Dept. of Microbiology. Center for Biofilm Engineering
+ Show Author Affiliations

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 Q2715%, Q3010%, and Q3215% for V1V2, V4, and V6, respectively, estimated similar values as obtained with clone libraries and Sanger sequencing. The most stringent Q tested (Q3210%) 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.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Science Foundation (NSF)
Grant/Contract Number:
AC02-05CH11231; DGE0654336
OSTI ID:
1816633
Journal Information:
Microbial Ecology, Vol. 64, Issue 2; ISSN 0095-3628
Publisher:
SpringerCopyright Statement
Country of Publication:
United States
Language:
English

References (18)

454 sequencing put to the test using the complex genome of barley journal October 2006
Microbial diversity in the deep sea and the underexplored "rare biosphere" journal July 2006
Ironing out the wrinkles in the rare biosphere through improved OTU clustering: Ironing out the wrinkles in the rare biosphere journal March 2010
Genome sequencing in microfabricated high-density picolitre reactors journal July 2005
Accuracy and quality of massively parallel DNA pyrosequencing journal January 2007
Bacterial community succession during in situ uranium bioremediation: spatial similarities along controlled flow paths journal September 2008
Short pyrosequencing reads suffice for accurate microbial community analysis journal August 2007
Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers journal September 2008
Accurate determination of microbial diversity from 454 pyrosequencing data journal August 2009
Insidious effects of sequencing errors on perceived diversity in molecular surveys: Letters journal September 2010
Species richness in soil bacterial communities: A proposed approach to overcome sample size bias journal September 2008
Massively parallel rRNA gene sequencing exacerbates the potential for biased community diversity comparisons due to variable library sizes: Pyrosequencing exacerbates sample size bias journal September 2011
Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons journal January 2011
Compilation of small ribosomal subunit RNA sequences journal April 1991
Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates journal January 2010
Confidence intervals of similarity values determined for cloned SSU rRNA genes from environmental samples journal April 2006
Compilation of small ribosomal subunit RNA sequences journal April 1990
DNA recovery from soils of diverse composition. journal January 1996

Cited By (10)

Microbial community changes during a toxic cyanobacterial bloom in an alkaline Hungarian lake journal August 2018
Direct 16S rRNA-seq from bacterial communities: a PCR-independent approach to simultaneously assess microbial diversity and functional activity potential of each taxon journal August 2016
Mineral Microniches Control the Diversity of Subsurface Microbial Populations journal January 2014
Molecular study of bacterial diversity within the trophosome of the vestimentiferan tubeworm Ridgeia piscesae journal July 2014
Improved taxonomic assignment of human intestinal 16S rRNA sequences by a dedicated reference database journal December 2015
Phylogenetic Affiliation of SSU rRNA Genes Generated by Massively Parallel Sequencing: New Insights into the Freshwater Protist Diversity journal March 2013
A Lipid-Accumulating Alga Maintains Growth in Outdoor, Alkaliphilic Raceway Pond with Mixed Microbial Communities journal January 2016
Exploring Biodiversity and Arsenic Metabolism of Microbiota Inhabiting Arsenic-Rich Groundwaters in Northern Italy journal July 2019
Influence of Artifact Removal on Rare Species Recovery in Natural Complex Communities Using High-Throughput Sequencing journal May 2014
Archaeal and bacterial communities in three alkaline hot springs in Heart Lake Geyser Basin, Yellowstone National Park journal January 2013

Similar Records

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
species richness
clone library
species richness estimate
prime error
ambiguous nucleotide