Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

Marotz, Clarisse; Sharma, Anukriti; Humphrey, Greg; Gottel, Neil; Daum, Christopher; Gilbert, Jack A.; Eloe‐Fadrosh, Emiley A.; Knight, Rob

doi:10.2144/btn-2018-0192

Cited by 59 publications

(51 citation statements)

References 12 publications

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“…suggested that DNBSEQ-G400 was more stable than MiSeq. Good consistency among PCR replicates matched with previously reported results, mainly due to the substantial improvements in the processivity and fidelity of DNA polymerase (30). As such, PCR replicates seem to be unnecessary in future projects with possible budget constraints.…”

Section: The Evaluation Of Pcr and Run Effects On Sequencing Of Technsupporting

confidence: 84%

Efficient and stable metabarcoding sequencing from DNBSEQ-G400 sequencer examined by large fungal community analysis

Sun

Wang

Fang

et al. 2020

Preprint

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ABSTRACTMetabarcoding has become the de facto method for characterizing the structure of microbial communities in complex environmental samples. To determine how sequencing platform may influence microbial community characterization, we present a large-scale comparison of two sequencing platforms; Illumina MiSeq and a new platform DNBSEQ-G400 developed by MGI Tech. The accuracy of DNBSEQ-G400 on bacterial and fungal mock samples and compared sequencing consistency and precision between DNBSEQ-G400 and MiSeq platforms by sequencing the fungal ITS2 region from 1144 soil samples with 3 technical replicates. The DNBSEQ-G400 showed a high accuracy in reproducing mock communities containing different proportions of bacteria and fungi, respectively. The taxonomic profiles of the 1144 soil samples generated by the two DNBSEQ-G400 modes closely resembled each other and were highly correlated with those generated by the MiSeq platform. Analyses of technical replicates demonstrated a run bias against certain taxa on the MiSeq but not DNBSEQ-G400 platform. Based on lower cost, greater capacity, and less bias, we conclude that DNBSEQ-G400 is an optimal platform for short-term metabarcoding of microbial communities.IMPORTANCEExperimental steps that generate sequencing bias during amplicon sequencing have been intensively evaluated, including the choice of primer pair, polymerase, PCR cycle and technical replication. However, few studies have assessed the accuracy and precision of different sequencing platforms. Here, we compared the performance of newly released DNBSEQ-G400 sequencer with that of the commonly used Illumina MiSeq platform by leveraging amplicon sequencing of a large number of soil samples. Significant sequencing bias among major fungal genera was found in parallel MiSeq runs, which can be easily neglected without the use of sequencing controls. We emphasize the importance of technical controls in large-scale sequencing efforts and provide DNBSEQ-G400 as an alternative with increased sequencing capacity and more stable reproducibility for amplicon sequencing.

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Section: The Evaluation Of Pcr and Run Effects On Sequencing Of Technsupporting

confidence: 84%

Efficient and stable metabarcoding sequencing from DNBSEQ-G400 sequencer examined by large fungal community analysis

Sun

Wang

Fang

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…We confirmed the DNA yield, purity, and integrity for each extraction using a Qubit fluorometer, Nanodrop 1000 spectrophotometer, and agarose gel electrophoresis. For each sample ( 88 ), the hypervariable V4 region of the 16S rRNA gene was amplified using the universal Earth Microbiome Project primer pairs F515 and R806 ( 89 ). The amplicons were subjected to Illumina paired-end sequencing at the Australian Centre for Ecogenomics, University of Queensland.…”

Section: Methodsmentioning

confidence: 99%

Soil Bacterial Communities Exhibit Strong Biogeographic Patterns at Fine Taxonomic Resolution

et al. 2020

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Bacteria have been inferred to exhibit relatively weak biogeographic patterns. To what extent such findings reflect true biological phenomena or methodological artifacts remains unclear. Here, we addressed this question by analyzing the turnover of soil bacterial communities from three data sets. We applied three methodological innovations: (i) design of a hierarchical sampling scheme to disentangle environmental from spatial factors driving turnover; (ii) resolution of 16S rRNA gene amplicon sequence variants to enable higher-resolution community profiling; and (iii) application of the new metric zeta diversity to analyze multisite turnover and drivers. At fine taxonomic resolution, rapid compositional turnover was observed across multiple spatial scales. Turnover was overwhelmingly driven by deterministic processes and influenced by the rare biosphere. The communities also exhibited strong distance decay patterns and taxon-area relationships, with z values within the interquartile range reported for macroorganisms. These biogeographical patterns were weakened upon applying two standard approaches to process community sequencing data: clustering sequences at 97% identity threshold and/or filtering the rare biosphere (sequences lower than 0.05% relative abundance). Comparable findings were made across local, regional, and global data sets and when using shotgun metagenomic markers. Altogether, these findings suggest that bacteria exhibit strong biogeographic patterns, but these signals can be obscured by methodological limitations. We advocate various innovations, including using zeta diversity, to advance the study of microbial biogeography. IMPORTANCE It is commonly thought that bacterial distributions show lower spatial variation than for multicellular organisms. In this article, we present evidence that these inferences are artifacts caused by methodological limitations. Through leveraging innovations in sampling design, sequence processing, and diversity analysis, we provide multifaceted evidence that bacterial communities in fact exhibit strong distribution patterns. This is driven by selection due to factors such as local soil characteristics. Altogether, these findings suggest that the processes underpinning diversity patterns are more unified across all domains of life than previously thought, which has broad implications for the understanding and management of soil biodiversity.

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“…The PCR reactions were prepared in a pre-PCR laboratory in a 5% bleached-cleaned and UV irradiated hood. Single reactions [35] of 2.5 μL X10 HiFi buffer, 0.1 μL Platinum™ Taq DNA Polymerase (ThermoFisher), 19.2 μL dH2O, 0.2 μL 100 mM dNTP mix, 0.5 μL each of 10 μM forward and reverse primer and 1 μL DNA. DNA was amplified using an initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 1 min, elongation at 68°C for 90 s, with final adenylation for 10 min at 68°C, in line with the Earth Microbiome Protocol [36].…”

Section: Amplicon Library Preparation Quantification and Dna Sequenmentioning

confidence: 99%

Signatures of landscape and captivity in the gut microbiota of Southern Hairy-nosed Wombats (Lasiorhinus latifrons)

2021

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Background Herbivorous mammals co-opt microbes to derive energy and nutrients from diets that are recalcitrant to host enzymes. Recent research has found that captive management—an important conservation tool for many species—can alter the gut microbiota of mammals. Such changes could negatively impact the ability of herbivorous mammals to derive energy from their native diets, and ultimately reduce host fitness. To date, nothing is known of how captivity influences the gut microbiota of the Southern Hairy-nosed Wombat (SHNW), a large herbivorous marsupial that inhabits South Australia. Here, using 16S rRNA gene sequencing, we characterized the faecal microbiota of SHNWs in captivity and from three wild populations, two from degraded habitats and one from an intact native grass habitat. Results We found that captive SHNWs had gut microbiota that were compositionally different and less diverse compared to wild SHNWs. There were major differences in gut microbiota community membership between captive and wild animals, both in statistically significant changes in relative abundance of microbes, and in the presence/absence of microbes. We also observed differences in microbial composition between wild populations, with the largest difference associated with native vs. degraded habitat. Conclusions These results suggest that captivity has a major impact on the gut microbiota of SHNWs, and that different wild populations harbour distinct microbial compositions. Such findings warrant further work to determine what impacts these changes have on the fitness of SHNWs, and whether they could be manipulated to improve future management of the species.

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Triplicate PCR Reactions for 16S rRNA Gene Amplicon Sequencing are Unnecessary

Cited by 59 publications

References 12 publications

Efficient and stable metabarcoding sequencing from DNBSEQ-G400 sequencer examined by large fungal community analysis

Efficient and stable metabarcoding sequencing from DNBSEQ-G400 sequencer examined by large fungal community analysis

Soil Bacterial Communities Exhibit Strong Biogeographic Patterns at Fine Taxonomic Resolution

Signatures of landscape and captivity in the gut microbiota of Southern Hairy-nosed Wombats (Lasiorhinus latifrons)

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