Dataset Information

Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

ABSTRACT: Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first mined and then used to search for candidate primers within the fragments by measuring the coverage rate defined as the percentage of bacterial sequences containing the target. Thirty predicted primers with a high coverage rate (>90%) were identified, which were basically located in the same conservative regions as known primers in previous reports, whereas 30% of the known primers were associated with a coverage rate of <90%. The application scope of the primers was also examined by calculating the percentages of failed detections in bacterial phyla. Primers A519-539, E969-983, E1063-1081, U515 and E517, are highly recommended because of their high coverage in almost all phyla. As expected, the three predominant phyla, Firmicutes, Gemmatimonadetes and Proteobacteria, are best covered by the predicted primers. The primers recommended in this report shall facilitate a comprehensive and reliable survey of bacterial diversity in metagenomic studies.

SUBMITTER: Wang Y

PROVIDER: S-EPMC2754607 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

Wang Yong Y Qian Pei-Yuan PY

PloS one 20091009 10

Bacterial 16S ribosomal DNA (rDNA) amplicons have been widely used in the classification of uncultured bacteria inhabiting environmental niches. Primers targeting conservative regions of the rDNAs are used to generate amplicons of variant regions that are informative in taxonomic assignment. One problem is that the percentage coverage and application scope of the primers used in previous studies are largely unknown. In this study, conservative fragments of available rDNA sequences were first min ...[more]

PMID: 19816594

Similar Datasets

Project description:Sourdoughs (SDs) are spontaneously formed microbial ecosystems composed of various species of lactic acid bacteria (LAB) and acid-tolerant yeasts in food matrices of cereal flours mixed with water. To date, more than 90 LAB species have been isolated, significantly impacting the organoleptic characteristics, shelf life, and health properties of bakery products. To learn more about the unique bacterial communities involved in creating regional Bulgarian sourdoughs, we examined the metacommunities of five sourdoughs produced by spontaneous fermentation and maintained by backslopping in bakeries from three geographic locations. The 16S rRNA gene amplicon sequencing showed that the former genus Lactobacillus was predominant in the studied sourdoughs (51.0-78.9%). Weissella (0.9-42.8%), Herbaspirillum (1.6-3.8%), Serratia (0.1-11.7%), Pediococcus (0.2-7.5%), Bacteroides (0.1-1.3%), and Sphingomonas (0.1-0.5%) were also found in all 5 samples. Genera Leuconostoc, Enterococcus, Bacillus, and Asaia were sample-specific. It is interesting to note that the genus Weissella was more abundant in wholegrain samples. The greatest diversity at the species level was found in the former genus Lactobacillus, presented in the sourdough samples with 13 species. The UPGMA cluster analysis clearly demonstrated similarity in species' relative abundance between samples from the same location. In addition, we can conclude that the presence of two main clusters-one including samples from mountainous places (the cities of Smolyan and Bansko) and the other including samples from the city of Ruse (the banks of the Danube River)-may indicate the impact of climate and geographic location (e.g., terrain, elevation, land use, and nearby water bodies and their streams) on the abundance of microbiome taxa. As the bacterial population is crucial for bread standardization, we expect the local bakery sector to be interested in the relationship between process variables and their effect on bacterial dynamics described in this research study.

Project description:BACKGROUND:The 16S rRNA gene-based amplicon sequencing analysis is widely used to determine the taxonomic composition of microbial communities. Once the taxonomic composition of each community is obtained, evolutionary relationships among taxa are inferred by a phylogenetic tree. Thus, the combined representation of taxonomic composition and phylogenetic relationships among taxa is a powerful method for understanding microbial community structure; however, applying phylogenetic tree-based representation with information on the abundance of thousands or more taxa in each community is a difficult task. For this purpose, we previously developed the tool VITCOMIC (VIsualization tool for Taxonomic COmpositions of MIcrobial Community), which is based on the genome-sequenced microbes' phylogenetic information. Here, we introduce VITCOMIC2, which incorporates substantive improvements over VITCOMIC that were necessary to address several issues associated with 16S rRNA gene-based analysis of microbial communities. RESULTS:We developed VITCOMIC2 to provide (i) sequence identity searches against broad reference taxa including uncultured taxa; (ii) normalization of 16S rRNA gene copy number differences among taxa; (iii) rapid sequence identity searches by applying the graphics processing unit-based sequence identity search tool CLAST; (iv) accurate taxonomic composition inference and nearly full-length 16S rRNA gene sequence reconstructions for metagenomic shotgun sequencing; and (v) an interactive user interface for simultaneous representation of the taxonomic composition of microbial communities and phylogenetic relationships among taxa. We validated the accuracy of processes (ii) and (iv) by using metagenomic shotgun sequencing data from a mock microbial community. CONCLUSIONS:The improvements incorporated into VITCOMIC2 enable users to acquire an intuitive understanding of microbial community composition based on the 16S rRNA gene sequence data obtained from both metagenomic shotgun and amplicon sequencing.

Project description:Short-amplicon 16S rRNA gene sequencing is currently the method of choice for studies investigating microbiomes. However, comparative studies on differences in procedures are scarce. We sequenced human stool samples and mock communities with increasing complexity using a variety of commonly used protocols. Short amplicons targeting different variable regions (V-regions) or ranges thereof (V1-V2, V1-V3, V3-V4, V4, V4-V5, V6-V8, and V7-V9) were investigated for differences in the composition outcome due to primer choices. Next, the influence of clustering (operational taxonomic units [OTUs], zero-radius OTUs [zOTUs], and amplicon sequence variants [ASVs]), different databases (GreenGenes, the Ribosomal Database Project, Silva, the genomic-based 16S rRNA Database, and The All-Species Living Tree), and bioinformatic settings on taxonomic assignment were also investigated. We present a systematic comparison across all typically used V-regions using well-established primers. While it is known that the primer choice has a significant influence on the resulting microbial composition, we show that microbial profiles generated using different primer pairs need independent validation of performance. Further, comparing data sets across V-regions using different databases might be misleading due to differences in nomenclature (e.g., Enterorhabdus versus Adlercreutzia) and varying precisions in classification down to genus level. Overall, specific but important taxa are not picked up by certain primer pairs (e.g., Bacteroidetes is missed using primers 515F-944R) or due to the database used (e.g., Acetatifactor in GreenGenes and the genomic-based 16S rRNA Database). We found that appropriate truncation of amplicons is essential and different truncated-length combinations should be tested for each study. Finally, specific mock communities of sufficient and adequate complexity are highly recommended.IMPORTANCE In 16S rRNA gene sequencing, certain bacterial genera were found to be underrepresented or even missing in taxonomic profiles when using unsuitable primer combinations, outdated reference databases, or inadequate pipeline settings. Concerning the last, quality thresholds as well as bioinformatic settings (i.e., clustering approach, analysis pipeline, and specific adjustments such as truncation) are responsible for a number of observed differences between studies. Conclusions drawn by comparing one data set to another (e.g., between publications) appear to be problematic and require independent cross-validation using matching V-regions and uniform data processing. Therefore, we highlight the importance of a thought-out study design including sufficiently complex mock standards and appropriate V-region choice for the sample of interest. The use of processing pipelines and parameters must be tested beforehand.

Dataset Information

Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

Publications

Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets