UNOISE2: improved error-correction for Illumina 16S and ITS amplicon sequencing

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2016, Номер unknown

Опубликована: Окт. 15, 2016

Abstract Amplicon sequencing of tags such as 16S and ITS ribosomal RNA is a popular method for investigating microbial populations. In experiments, sequence errors caused by PCR are difficult to distinguish from true biological variation. I describe UNOISE2, an updated version the UNOISE algorithm denoising (error-correcting) Illumina amplicon reads show that it has comparable or better accuracy than DADA2.

Язык: Английский

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An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform

Microbiome, Год журнала: 2014, Номер 2(1)

Опубликована: Фев. 24, 2014

To take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development improved methods overcome technical limitations inherent platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on Illumina MiSeq platform and generates sequences suboptimal quality.Here we present an dual-indexing amplification approach assess communities from clinical samples using V3-V4 region gene platform. We introduced a 0 7 bp "heterogeneity spacer" index that allows equal proportion be sequenced out phase.Our yields high quality data both 250 300 paired-end protocols provides flexible cost-effective option.

Язык: Английский

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Soil bacterial networks are less stable under drought than fungal networks

Nature Communications, Год журнала: 2018, Номер 9(1)

Опубликована: Июль 27, 2018

Abstract Soil microbial communities play a crucial role in ecosystem functioning, but it is unknown how co-occurrence networks within these respond to disturbances such as climate extremes. This represents an important knowledge gap because changes could have implications for their functioning and vulnerability future disturbances. Here, we show grassland mesocosms that drought promotes destabilising properties soil bacterial, not fungal, networks, bacterial link more strongly during recovery than do fungal communities. Moreover, reveal has prolonged effect on via vegetation composition resultant reductions moisture. Our results provide new insight the mechanisms through which alters with potential long-term consequences, including plant community ability of aboveground belowground withstand

Язык: Английский

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Development of a Prokaryotic Universal Primer for Simultaneous Analysis of Bacteria and Archaea Using Next-Generation Sequencing

PLoS ONE, Год журнала: 2014, Номер 9(8), С. e105592 - e105592

Опубликована: Авг. 21, 2014

For the analysis of microbial community structure based on 16S rDNA sequence diversity, sensitive and robust PCR amplification is a critical step. To obtain accurate composition data, must be free bias; however, amplifying all species with equal efficiency from sample containing large variety microorganisms remains challenging. Here, we designed universal primer V3-V4 hypervariable region prokaryotic for simultaneous detection Bacteria Archaea in fecal samples crossbred pigs (Landrace × Large white Duroc) using an Illumina MiSeq next-generation sequencer. In-silico showed that newly primers matched approximately 98.0% 94.6% rRNA gene sequences Ribosomal Database Project database. each sequencing reaction performed primer, average 69,330 (± 20,482) reads were obtained, which archaeal genes comprised 1.2% to 3.2% reads. In addition, frequency belonging phylum Verrucomicrobia, including members classes Verrucomicrobiae Opitutae, was higher NGS than bacterial primer. Importantly, this new set had markedly lower bias most previously primers. Our findings demonstrate present study will permit Archaea, therefore allow more comprehensive understanding structures environmental samples.

Язык: Английский

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Error filtering, pair assembly and error correction for next-generation sequencing reads

Bioinformatics, Год журнала: 2015, Номер 31(21), С. 3476 - 3482

Опубликована: Июль 2, 2015

Abstract Motivation: Next-generation sequencing produces vast amounts of data with errors that are difficult to distinguish from true biological variation when coverage is low. Results: We demonstrate large reductions in error frequencies, especially for high-error-rate reads, by three independent means: (i) filtering reads according their expected number errors, (ii) assembling overlapping read pairs and (iii) amplicon exploiting unique sequence abundances perform correction. also show most published paired assemblers calculate incorrect posterior quality scores. Availability implementation: These methods implemented the USEARCH package. Binaries freely available at http://drive5.com/usearch. Contact: [email protected] Supplementary information: Bioinformatics online.

Язык: Английский

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Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation

Nature, Год журнала: 2016, Номер 533(7604), С. 543 - 546

Опубликована: Май 3, 2016

Our intestinal microbiota harbours a diverse bacterial community required for our health, sustenance and wellbeing. Intestinal colonization begins at birth climaxes with the acquisition of two dominant groups strict anaerobic bacteria belonging to Firmicutes Bacteroidetes phyla. Culture-independent, genomic approaches have transformed understanding role human microbiome in health many diseases. However, owing prevailing perception that indigenous are largely recalcitrant culture, their functions phenotypes remain unknown. Here we describe novel workflow based on targeted phenotypic culturing linked large-scale whole-genome sequencing, phylogenetic analysis computational modelling demonstrates substantial proportion culturable. Applying this approach healthy individuals, isolated 137 species from characterized candidate families, genera were archived as pure cultures. Whole-genome metagenomic combined analysis, suggests least 50-60% individual produce resilient spores, specialized host-to-host transmission. unlocks reveals how marked oxygen-sensitive can be transmitted between affecting heritability.

Язык: Английский

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Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy

Science, Год журнала: 2020, Номер 369(6510), С. 1481 - 1489

Опубликована: Авг. 13, 2020

Several species of intestinal bacteria have been associated with enhanced efficacy checkpoint blockade immunotherapy, but the underlying mechanisms by which microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species-Bifidobacterium pseudolongum, Lactobacillus johnsonii, and Olsenella species-that significantly immune inhibitors in four mouse models cancer. We found that B. pseudolongum modulated immunotherapy response through production metabolite inosine. Decreased gut barrier function induced increased systemic translocation inosine activated T cells. The effect was dependent on cell expression adenosine A2A receptor required costimulation. Collectively, our study identifies a previously unknown microbial pathway may be exploited to develop microbial-based adjuvant therapies.

Язык: Английский

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Quantitative microbiome profiling links gut community variation to microbial load

Nature, Год журнала: 2017, Номер 551(7681), С. 507 - 511

Опубликована: Ноя. 1, 2017

Язык: Английский

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Emergent simplicity in microbial community assembly

Science, Год журнала: 2018, Номер 361(6401), С. 469 - 474

Опубликована: Авг. 3, 2018

Interchanging species of similar function Under natural conditions, bacteria form mixed, interacting communities. Understanding how such communities assemble and stabilize is important in a range contexts, from biotechnological applications to what happens our guts. Goldford et al. sampled the microbial soil plants containing hundreds thousands sequence variants. The organisms were passaged after culture low concentrations single carbon sources cross-fed with each other's metabolites; then, resulting sequenced using 16S ribosomal RNA, outcomes modeled mathematically. mix that survived under steady conditions converged reproducibly reflect experimentally imposed rather than initially inoculated—although at coarse phylogenetic levels, taxonomic patterns persisted. Science , this issue p. 469

Язык: Английский

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SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2016, Номер unknown

Опубликована: Сен. 9, 2016

Abstract Metagenomics experiments often characterize microbial communities by sequencing the ribosomal 16S and ITS regions. Taxonomy prediction is a fundamental step in such studies. The SINTAX algorithm predicts taxonomy using k -mer similarity to identify top hit reference database provides bootstrap confidence for all ranks prediction. achieves comparable or better accuracy RDP Naive Bayesian Classifier with simpler that does not require training. Most tested methods are shown have high rates of over-classification errors where novel taxa incorrectly predicted known names.

Язык: Английский

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