Ribosomal Database Project: data and tools for high throughput rRNA analysis

Nucleic Acids Research, Год журнала: 2013, Номер 42(D1), С. D633 - D642

Опубликована: Ноя. 27, 2013

Ribosomal Database Project (RDP; http://rdp.cme.msu.edu/) provides the research community with aligned and annotated rRNA gene sequence data, along tools to allow researchers analyze their own sequences in RDP framework. data are utilized fields as diverse human health, microbial ecology, environmental microbiology, nucleic acid chemistry, taxonomy phylogenetics. In addition collections of bacterial archaeal small subunit genes, now includes a collection fungal large genes. tools, including Classifier Aligner, have been updated work this new collection. The use high-throughput sequencing characterize populations has exploded past several years, technologies improved, sizes datasets increased. With release 11, is providing an expanded set facilitate analysis both single-stranded paired-end reads. addition, most available open source packages for download local by high-volume needs or who would like develop custom pipelines.

Язык: Английский

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Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients

Science, Год журнала: 2017, Номер 359(6371), С. 97 - 103

Опубликована: Ноя. 2, 2017

Preclinical mouse models suggest that the gut microbiome modulates tumor response to checkpoint blockade immunotherapy; however, this has not been well-characterized in human cancer patients. Here we examined oral and of melanoma patients undergoing anti-programmed cell death 1 protein (PD-1) immunotherapy (

Язык: Английский

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The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update

Nucleic Acids Research, Год журнала: 2018, Номер 46(W1), С. W537 - W544

Опубликована: Май 3, 2018

Galaxy (homepage: https://galaxyproject.org, main public server: https://usegalaxy.org) is a web-based scientific analysis platform used by tens of thousands scientists across the world to analyze large biomedical datasets such as those found in genomics, proteomics, metabolomics and imaging. Started 2005, continues focus on three key challenges data-driven science: making analyses accessible all researchers, ensuring are completely reproducible, it simple communicate so that they can be reused extended. During last two years, team open-source community around have made substantial improvements Galaxy's core framework, user interface, tools, training materials. Framework interface now enable for analyzing datasets, >5500 tools available from ToolShed. The has led an effort create numerous high-quality tutorials focused common types genomic analyses. developer communities continue grow integral development. number servers, developers contributing framework its users server increased substantially.

Язык: Английский

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Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

Genome Research, Год журнала: 2011, Номер 21(3), С. 494 - 504

Опубликована: Янв. 6, 2011

Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric amplification products. Chimeras are hybrid products between multiple parent sequences that falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras greater sensitivity than previous methods, performs well on short such those produced the 454 Life Sciences (Roche) Genome Sequencer, scale to large data sets. By benchmarking performance against derived from controlled DNA mixture known organisms simulated set, we provide insights into factors affect sequence abundance, extent similarity genes, PCR conditions. were found reproducibly form independent amplifications contributed false perceptions identification taxa, less-abundant species exhibiting rates exceeding 70%. Shotgun metagenomic our mock community appear devoid chimeras, supporting role for shotgun metagenomics in validating discovered targeted surveys.

Язык: Английский

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Towards a unified paradigm for sequence‐based identification of fungi

Molecular Ecology, Год журнала: 2013, Номер 22(21), С. 5271 - 5277

Опубликована: Авг. 3, 2013

Abstract The nuclear ribosomal internal transcribed spacer ( ITS ) region is the formal fungal barcode and in most cases marker of choice for exploration diversity environmental samples. Two problems are particularly acute pursuit satisfactory taxonomic assignment newly generated sequences: (i) lack an inclusive, reliable public reference data set (ii) means to refer species, which no Latin name available a standardized stable way. Here, we report on progress these regards through further development UNITE database http://unite.ut.ee molecular identification fungi. All species represented by at least two sequences international nucleotide sequence databases now given unique, accession number type (e.g. H ymenoscyphus pseudoalbidus | GU 586904| SH 133781.05 FU ), their ecological annotations were corrected as far possible distributed, third‐party annotation effort. We introduce term ‘species hypothesis’ taxa discovered clustering different similarity thresholds (97–99%). An automatically or manually designated chosen represent each such SH. These released http://unite.ut.ee/repository.php use scientific community in, example, local searches QIIME pipeline. system will be updated grows. invite everybody position improve metadata associated with particular lineages expertise do so new Web‐based management .

Язык: Английский

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Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples

Environmental Microbiology, Год журнала: 2015, Номер 18(5), С. 1403 - 1414

Опубликована: Авг. 14, 2015

Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated Gammaproteobacteria) common marine taxa. evaluated samples mock communities (containing 11 27 clones), showing alternative primers 515F-Y (5'-GTGYCAGCMGCCGCGGTAA) 926R (5'-CCGYCAATTYMTTTRAGTTT) yield more accurate estimates abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, amplify eukaryotic 18S rRNA. Mock 515F-Y/926R yielded closer observed composition versus expected (r(2) = 0.95) compared 515F-Y/806R ∼ 0.5). Unexpectedly, biases against SAR11 in field (∼4-10-fold) were stronger than (∼2-fold). Correcting a mismatch to Thaumarchaea 515F-C increased their apparent abundance samples, but not as much using rather 806R. With plankton rich DNA (> 1 μm size fraction), sequences averaged ∼17% all sequences. A single strongly bias amplification, even perfectly matched exhibit preferential amplification. show beyond silico predictions, testing important selection.

Язык: Английский

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SINA: Accurate high-throughput multiple sequence alignment of ribosomal RNA genes

Bioinformatics, Год журнала: 2012, Номер 28(14), С. 1823 - 1829

Опубликована: Май 3, 2012

Abstract Motivation: In the analysis of homologous sequences, computation multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like ribosomal RNA (rRNA) where already millions sequences are publicly available and individual studies can easily produce hundreds thousands new sequences. Methods have been developed to cope with such numbers, but further improvements needed meet accuracy requirements. Results: this study, we present SILVA Incremental Aligner (SINA) used align rRNA gene databases provided by project. SINA uses combination k-mer searching partial order alignment (POA) maintain very high while satisfying throughput performance demands. was evaluated in comparison commonly MSA programs PyNAST mothur. The three BRAliBase III benchmark MSAs could be reproduced 99.3, 97.6 96.1 accuracy. A larger comprising 38 772 98.9 99.3% using reference 1000 5000 able achieve higher than mothur all performed benchmarks. Availability: Alignment up 500 latest SSU/LSU Ref datasets as offered at http://www.arb-silva.de/aligner. page also links Linux binaries, user manual tutorial. made under personal use license. Contact: [email protected] Supplementary information: data Bioinformatics online.

Язык: Английский

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Reagent and laboratory contamination can critically impact sequence-based microbiome analyses

BMC Biology, Год журнала: 2014, Номер 12(1)

Опубликована: Ноя. 12, 2014

The study of microbial communities has been revolutionised in recent years by the widespread adoption culture independent analytical techniques such as 16S rRNA gene sequencing and metagenomics. One potential confounder these sequence-based approaches is presence contamination DNA extraction kits other laboratory reagents. In this we demonstrate that contaminating ubiquitous commonly used reagents, varies greatly composition between different kit batches, critically impacts results obtained from samples containing a low biomass. Contamination both PCR-based surveys shotgun We provide an extensive list genera, guidelines on how to mitigate effects contamination. These suggest caution should be advised when applying microbiota present biomass environments. Concurrent negative control strongly advised.

Язык: Английский

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The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications

Nucleic Acids Research, Год журнала: 2018, Номер 47(D1), С. D259 - D264

Опубликована: Окт. 12, 2018

UNITE (https://unite.ut.ee/) is a web-based database and sequence management environment for the molecular identification of fungi. It targets formal fungal barcode-the nuclear ribosomal internal transcribed spacer (ITS) region-and offers all ∼1 000 public ITS sequences reference. These are clustered into ∼459 species hypotheses assigned digital object identifiers (DOIs) to promote unambiguous reference across studies. In-house third-party curation annotation have resulted in more than 275 improvements data over past 15 years. serves as provider range metabarcoding software pipelines regularly exchanges with major databases other community resources. Recent include redesigned handling unclassifiable hypotheses, integration taxonomic backbone Global Biodiversity Information Facility, support an unlimited number parallel classification systems.

Язык: Английский

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Waste Not, Want Not: Why Rarefying Microbiome Data Is Inadmissible

PLoS Computational Biology, Год журнала: 2014, Номер 10(4), С. e1003531 - e1003531

Опубликована: Апрель 3, 2014

Current practice in the normalization of microbiome count data is inefficient statistical sense. For apparently historical reasons, common approach either to use simple proportions (which does not address heteroscedasticity) or rarefying counts, even though both these approaches are inappropriate for detection differentially abundant species. Well-established theory available that simultaneously accounts library size differences and biological variability using an appropriate mixture model. Moreover, specific implementations DNA sequencing read (based on a Negative Binomial model instance) already RNA-Seq focused R packages such as edgeR DESeq. Here we summarize supporting simulations empirical demonstrate substantial improvements provided by relevant framework over rarefying. We show how rarefied counts result high rate false positives tests species across sample classes. Regarding sample-wise clustering, also procedure often discards samples can be accurately clustered alternative methods. further compare different methods with recently-described zero-inflated Gaussian mixture, implemented package called metagenomeSeq. find metagenomeSeq performs well when there adequate number replicates, but it nevertheless tends toward higher positive rate. Based results well-established theory, advocate investigators avoid altogether. have microbiome-specific extensions tools package, phyloseq.

Язык: Английский

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