Full 16S rRNA for primer design
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ABSTRACT:
PROVIDER: PRJEB30777 | ENA |
REPOSITORIES: ENA
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Project description: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.
| S-EPMC2754607 | biostudies-literature
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.
| S-EPMC8544895 | biostudies-literature
Project description:Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6-V8, and V7-V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. Beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.
| S-EPMC4523815 | biostudies-literature
Project description:Bacterial taxonomic community analyses using PCR-amplification of the 16S rRNA gene and high-throughput sequencing has become a cornerstone in microbiology research. To reliably detect the members, or operational taxonomic units (OTUs), that make up bacterial communities, taxonomic surveys rely on the use of the most informative PCR primers to amplify the broad range of phylotypes present in up-to-date reference databases. However, primers specific for the domain Bacteria were often developed some time ago against database versions that are now out of date. Here we evaluated the performance of four bacterial primers for characterizing complex microbial communities in explosives contaminated and non-contaminated forest soil and by in silico evaluation against the current SILVA123 database. Primer pair 341f/785r produced the highest number of bacterial OTUs, phylogenetic richness, Shannon diversity, low non-specificity and most reproducible results, followed by 967f/1391r and 799f/1193r. Primer pair 68f/518r showed overall low coverage and a bias toward Alphaproteobacteria. In silico, primer pair 341f/785r showed the highest coverage of the domain Bacteria (96.1%) with no obvious bias toward the majority of bacterial species. This suggests the high utility of primer pair 341f/785r for soil and plant-associated bacterial microbiome studies.
| S-EPMC5368227 | biostudies-literature
Project description:Evaluating Primer Bias in Full-Length 16S Sequencing of Synthetic and Canine Fecal Samples
| PRJEB85420 | ENA
Project description:BackgroundThe full-length 16S rRNA sequencing can better improve the taxonomic and phylogenetic resolution compared to the partial 16S rRNA gene sequencing. The 16S-FAS-NGS (16S rRNA full-length amplicon sequencing based on a next-generation sequencing platform) technology can generate high-quality, full-length 16S rRNA gene sequences using short-read sequencers, together with assembly procedures. However there is a lack of a data analysis suite that can help process and analyze the synthetic long read data.ResultsHerein, we developed software named 16S-FASAS (16S full-length amplicon sequencing data analysis software) for 16S-FAS-NGS data analysis, which provided high-fidelity species-level microbiome data. 16S-FASAS consists of data quality control, de novo assembly, annotation, and visualization modules. We verified the performance of 16S-FASAS on both mock and fecal samples. In mock communities, we proved that taxonomy assignment by MegaBLAST had fewer misclassifications and tended to find more low abundance species than the USEARCH-UNOISE3-based classifier, resulting in species-level classification of 85.71% (6/7), 85.71% (6/7), 72.72% (8/11), and 70% (7/10) of the target bacteria. When applied to fecal samples, we found that the 16S-FAS-NGS datasets generated contigs grouped into 60 and 56 species, from which 71.62% (43/60) and 76.79% (43/56) were shared with the Pacbio datasets.Conclusions16S-FASAS is a valuable tool that helps researchers process and interpret the results of full-length 16S rRNA gene sequencing. Depending on the full-length amplicon sequencing technology, the 16S-FASAS pipeline enables a more accurate report on the bacterial complexity of microbiome samples. 16S-FASAS is freely available for use at https://github.com/capitalbio-bioinfo/FASAS.
| S-EPMC9511998 | biostudies-literature
Project description:The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.
| S-EPMC3989492 | biostudies-literature
Project description:This study evaluated the ammonium oxidizing communities (COA) associated with a potato crop (Solanum phureja) rhizosphere soil in the savannah of Bogotá (Colombia) by examining the presence and abundance of amoA enzyme genes and transcripts by qPCR and next-generation sequence analysis. amoA gene abundance could not be quantified by qPCR due to problems inherent in the primers; however, the melting curve analysis detected increased fluorescence for Bacterial communities but not for Archaeal communities. Transcriptome analysis by next-generation sequencing revealed that the majority of reads mapped to ammonium-oxidizing Archaea, suggesting that this activity is primarily governed by the microbial group of the Crenarchaeota phylum. In contrast,a lower number of reads mapped to ammonia-oxidizing bacteria.
2018-08-06 | GSE71824 | GEO
Project description:Describing the microbial community within the tumour has been a key aspect in understanding the pathophysiology of the tumour microenvironment. In head and neck cancer (HNC), most studies on tissue samples have only performed 16S rRNA short-read sequencing (SRS) on V3-V5 region. SRS is mostly limited to genus level identification. In this study, we compared full-length 16S rRNA long-read sequencing (FL-ONT) from Oxford Nanopore Technology (ONT) to V3-V4 Illumina SRS (V3V4-Illumina) in 26 HNC tumour tissues. Further validation was also performed using culture-based methods in 16 bacterial isolates obtained from 4 patients using MALDI-TOF MS. We observed similar alpha diversity indexes between FL-ONT and V3V4-Illumina. However, beta-diversity was significantly different between techniques (PERMANOVA - R2 = 0.131, p < 0.0001). At higher taxonomic levels (Phylum to Family), all metrics were more similar among sequencing techniques, while lower taxonomy displayed more discrepancies. At higher taxonomic levels, correlation in relative abundance from FL-ONT and V3V4-Illumina were higher, while this correlation decreased at lower levels. Finally, FL-ONT was able to identify more isolates at the species level that were identified using MALDI-TOF MS (75% vs. 18.8%). FL-ONT was able to identify lower taxonomic levels at a better resolution as compared to V3V4-Illumina 16S rRNA sequencing.
| S-EPMC11076400 | biostudies-literature
Project description:Evaluating Primer Bias in Full-Length 16S Sequencing of Human Fecal Samples
| PRJEB85714 | ENA