16S rRNA amplicon sequencing of salivary and esophageal microbiota using Illumina MiSeq
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB46880 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR6458290_1.fastq.gz | Fastqsanger.gz | |||
| ERR6458290_2.fastq.gz | Fastqsanger.gz | |||
| ERR6458300_1.fastq.gz | Fastqsanger.gz | |||
| ERR6458300_2.fastq.gz | Fastqsanger.gz | |||
| ERR6458310_1.fastq.gz | Fastqsanger.gz |
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Project description:16S rRNA amplicon sequencing of salivary and esophageal microbiota using PacBio
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Project description:BackgroundThe development of high-throughput sequencing technologies has revolutionized the field of microbial ecology via the sequencing of phylogenetic marker genes (e.g. 16S rRNA gene amplicon sequencing). Denoising, the removal of sequencing errors, is an important step in preprocessing amplicon sequencing data. The increasing popularity of the Illumina MiSeq platform for these applications requires the development of appropriate denoising methods.ResultsThe newly proposed denoising algorithm IPED includes a machine learning method which predicts potentially erroneous positions in sequencing reads based on a combination of quality metrics. Subsequently, this information is used to group those error-containing reads with correct reads, resulting in error-free consensus reads. This is achieved by masking potentially erroneous positions during this clustering step. Compared to the second best algorithm available, IPED detects double the amount of errors. Reducing the error rate had a positive effect on the clustering of reads in operational taxonomic units, with an almost perfect correspondence between the number of clusters and the theoretical number of species present in the mock communities.ConclusionOur algorithm IPED is a powerful denoising tool for correcting sequencing errors in Illumina MiSeq 16S rRNA gene amplicon sequencing data. Apart from significantly reducing the error rate of the sequencing reads, it has also a beneficial effect on their clustering into operational taxonomic units. IPED is freely available at http://science.sckcen.be/en/Institutes/EHS/MCB/MIC/Bioinformatics/ .
| S-EPMC4850673 | biostudies-literature
Project description:The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.
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Project description: Not available
| S-EPMC11655651 | biostudies-literature
Project description:BackgroundTo take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality.ResultsHere we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp "heterogeneity spacer" to the index sequence that allows an equal proportion of samples to be sequenced out of phase.ConclusionsOur approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.
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Project description:Tissue microRNAs (miRNAs) can detect cancers and predict prognosis. Several recent studies reported that tissue, plasma, and saliva miRNAs share similar expression profiles. In this study, we investigated the diagnostic value of salivary miRNAs (including whole saliva and saliva supernatant) for detection of esophageal cancer. By Agilent microarray, six deregulated miRNAs from whole saliva samples from seven patients with esophageal cancer and three healthy controls were selected. The six selected miRNAs were subjected to validation of their expression levels by RT-qPCR using both whole saliva and saliva supernatant samples from an independent set of 39 patients with esophageal cancer and 19 healthy controls.
2012-10-03 | E-GEOD-41268 | biostudies-arrayexpress
Project description:Tissue microRNAs (miRNAs) can detect cancers and predict prognosis. Several recent studies reported that tissue, plasma, and saliva miRNAs share similar expression profiles. In this study, we investigated the diagnostic value of salivary miRNAs (including whole saliva and saliva supernatant) for detection of esophageal cancer.
2012-10-03 | GSE41268 | GEO
Project description:BACKGROUND:The bacterial 16S rRNA gene has historically been used in defining bacterial taxonomy and phylogeny. However, there are currently no high-throughput methods to sequence full-length 16S rRNA genes present in a sample with precision. RESULTS:We describe a method for sequencing near full-length 16S rRNA gene amplicons using the high throughput Illumina MiSeq platform and test it using DNA from human skin swab samples. Proof of principle of the approach is demonstrated, with the generation of 1,604 sequences greater than 1,300 nt from a single Nano MiSeq run, with accuracy estimated to be 100-fold higher than standard Illumina reads. The reads were chimera filtered using information from a single molecule dual tagging scheme that boosts the signal available for chimera detection. CONCLUSIONS:This method could be scaled up to generate many thousands of sequences per MiSeq run and could be applied to other sequencing platforms. This has great potential for populating databases with high quality, near full-length 16S rRNA gene sequences from under-represented taxa and environments and facilitates analyses of microbial communities at higher resolution.
| S-EPMC5036073 | biostudies-literature
Project description:BackgroundRecent advancements in next-generation sequencing (NGS) technology have ushered in significant improvements in sequencing speed and data throughput, thereby enabling the simultaneous analysis of a greater number of samples within a single sequencing run. This technology has proven particularly valuable in the context of microbial community profiling, offering a powerful tool for characterizing the microbial composition at the species level within a given sample. This profiling process typically involves the sequencing of 16S ribosomal RNA (rRNA) gene fragments. By scaling up the analysis to accommodate a substantial number of samples, sometimes as many as 2,000, it becomes possible to achieve cost-efficiency and minimize the introduction of potential batch effects. Our study was designed with the primary objective of devising an approach capable of facilitating the comprehensive analysis of 1,711 samples sourced from diverse origins, including oropharyngeal swabs, mouth cavity swabs, dental swabs, and human fecal samples. This analysis was based on data obtained from 16S rRNA metagenomic sequencing conducted on the Illumina MiSeq and HiSeq sequencing platforms.ResultsWe have designed a custom set of 10-base pair indices specifically tailored for the preparation of libraries from amplicons derived from the V3-V4 region of the 16S rRNA gene. These indices are instrumental in the analysis of the microbial composition in clinical samples through sequencing on the Illumina MiSeq and HiSeq platforms. The utilization of our custom index set enables the consolidation of a significant number of libraries, enabling the efficient sequencing of these libraries in a single run.ConclusionsThe unique array of 10-base pair indices that we have developed, in conjunction with our sequencing methodology, will prove highly valuable to laboratories engaged in sequencing on Illumina platforms or utilizing Illumina-compatible kits.
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Project description:Phytophthora ITS1 amplicon Illumina MiSeq sequencing in tree nurseries and wider environment.
| PRJEB76241 | ENA