Dataset Information

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets.

ABSTRACT: As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on ?- and ?-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3-V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in ?-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based ?- and ?-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions.

SUBMITTER: Pinto AJ

PROVIDER: S-EPMC3419673 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets.

Pinto Ameet J AJ Raskin Lutgarde L

PloS one 20120815 8

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively ...[more]

PMID: 22905208

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Project description:BackgroundSequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention.ResultsIn this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon's index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (P<0.01), but not between nested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance <0.167) accounted for most of the distortion (>27% of total OTUs in stool).ConclusionsNested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies.

Project description:BACKGROUND: Soil bacteria are important drivers for nearly all biogeochemical cycles in terrestrial ecosystems and participate in most nutrient transformations in soil. In contrast to the importance of soil bacteria for ecosystem functioning, we understand little how different management types affect the soil bacterial community composition. METHODOLOGY/PRINCIPAL FINDINGS: We used pyrosequencing-based analysis of the V2-V3 16S rRNA gene region to identify changes in bacterial diversity and community structure in nine forest and nine grassland soils from the Schwäbische Alb that covered six different management types. The dataset comprised 598,962 sequences that were affiliated to the domain Bacteria. The number of classified sequences per sample ranged from 23,515 to 39,259. Bacterial diversity was more phylum rich in grassland soils than in forest soils. The dominant taxonomic groups across all samples (>1% of all sequences) were Acidobacteria, Alphaproteobacteria, Actinobacteria, Betaproteobacteria, Deltaproteobacteria, Gammaproteobacteria, and Firmicutes. Significant variations in relative abundances of bacterial phyla and proteobacterial classes, including Actinobacteria, Firmicutes, Verrucomicrobia, Cyanobacteria, Gemmatimonadetes and Alphaproteobacteria, between the land use types forest and grassland were observed. At the genus level, significant differences were also recorded for the dominant genera Phenylobacter, Bacillus, Kribbella, Streptomyces, Agromyces, and Defluviicoccus. In addition, soil bacterial community structure showed significant differences between beech and spruce forest soils. The relative abundances of bacterial groups at different taxonomic levels correlated with soil pH, but little or no relationships to management type and other soil properties were found. CONCLUSIONS/SIGNIFICANCE: Soil bacterial community composition and diversity of the six analyzed management types showed significant differences between the land use types grassland and forest. Furthermore, bacterial community structure was largely driven by tree species and soil pH.

Project description:Termites constitute part of diverse and economically important termite fauna in Africa, but information on gut microbiota and their associated soil microbiome is still inadequate. In this study, we assessed and compared the bacterial diversity and community structure between termites' gut, their mounds and surrounding soil using the 454 pyrosequencing-based analysis of 16S rRNA gene sequences. A wood-feeder termite (Microcerotermes sp.), three fungus-cultivating termites (Macrotermes michaelseni, Odontotermes sp. and Microtermes sp.), their associated mounds and corresponding savannah soil samples were analyzed. The pH of the gut homogenates and soil physico-chemical properties were determined. The results indicated significant difference in bacterial community composition and structure between the gut and corresponding soil samples. Soil samples (Chao1 index ranged from 1359 to 2619) had higher species richness than gut samples (Chao1 index ranged from 461 to 1527). The bacterial composition and community structure in the gut of Macrotermes michaelseni and Odontotermes sp. were almost identical but different from that of Microtermes and Microcerotermes species, which had unique community structures. The most predominant bacterial phyla in the gut were Bacteroidetes (40-58 %), Spirochaetes (10-70 %), Firmicutes (17-27 %) and Fibrobacteres (13 %) while in the soil samples were Acidobacteria (28-45 %), Actinobacteria (20-40 %) and Proteobacteria (18-24 %). Some termite gut-specific bacterial lineages belonging to the genera Dysgonomonas, Parabacteroides, Paludibacter, Tannerella, Alistipes, BCf9-17 termite group and Termite Treponema cluster were observed. The results not only demonstrated a high level of bacterial diversity in the gut and surrounding soil environments, but also presence of distinct bacterial communities that are yet to be cultivated. Therefore, combined efforts using both culture and culture-independent methods are suggested to comprehensively characterize the bacterial species and their specific roles in these environments.

Dataset Information

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets.

Publications

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets.

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