Multi-factorial examination of amplicon sequencing workflows from sample preparation to bioinformatic analysis

Wolfe, Travis J. De; Wright, Erik S.

doi:10.1186/s12866-023-02851-8

Cited by 9 publications

(2 citation statements)

References 77 publications

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“…16S library preparation ( 76 ) for the Muribaculaceae-depleted and -positive samples was performed at Gut4Health, BC Children’s Hospital Research Institute, Vancouver, BC, Canada. The V4 region was amplified using the following sequences: F: 5′- TATGGTAATTGTGTG YCAGCMGCCGCGGTAA and R: 5′- AGTCAGTCAGCCGGACTAC NVGGGTWTCTAAT.…”

Section: Methodsmentioning

confidence: 99%

PUPpy: a primer design pipeline for substrain-level microbial detection and absolute quantification

Ghezzi,

Fan,

et al. 2024

mSphere

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Characterizing microbial communities at high resolution and with absolute quantification is crucial to unravel the complexity and diversity of microbial ecosystems. This can be achieved with PCR assays, which enable highly selective detection and absolute quantification of microbial DNA. However, a major challenge that has hindered PCR applications in microbiome research is the design of highly specific primer sets that exclusively amplify intended targets. Here, we introduce Phylogenetically Unique Primers in python (PUPpy), a fully automated pipeline to design microbe- and group-specific primers within a given microbial community. PUPpy can be executed from a user-friendly graphical user interface, or two simple terminal commands, and it only requires coding sequence files of the community members as input. PUPpy-designed primers enable the detection of individual microbes and quantification of absolute microbial abundance in defined communities below the strain level. We experimentally evaluated the performance of PUPpy-designed primers using two bacterial communities as benchmarks. Each community comprises 10 members, exhibiting a range of genetic similarities that spanned from different phyla to substrains. PUPpy-designed primers also enable the detection of groups of bacteria in an undefined community, such as the detection of a gut bacterial family in a complex stool microbiota sample. Taxon-specific primers designed with PUPpy showed 100% specificity to their intended targets, without unintended amplification, in each community tested. Lastly, we show the absolute quantification of microbial abundance using PUPpy-designed primers in droplet digital PCR, benchmarked against 16S rRNA and shotgun sequencing. Our data shows that PUPpy-designed microbe-specific primers can be used to quantify substrain-level absolute counts, providing more resolved and accurate quantification in defined communities than short-read 16S rRNA and shotgun sequencing. IMPORTANCE Profiling microbial communities at high resolution and with absolute quantification is essential to uncover hidden ecological interactions within microbial ecosystems. Nevertheless, achieving resolved and quantitative investigations has been elusive due to methodological limitations in distinguishing and quantifying highly related microbes. Here, we describe Phylogenetically Unique Primers in python (PUPpy), an automated computational pipeline to design taxon-specific primers within defined microbial communities. Taxon-specific primers can be used to selectively detect and quantify individual microbes and larger taxa within a microbial community. PUPpy achieves substrain-level specificity without the need for computationally intensive databases and prioritizes user-friendliness by enabling both terminal and graphical user interface applications. Altogether, PUPpy enables fast, inexpensive, and highly accurate perspectives into microbial ecosystems, supporting the characterization of bacterial communities in both in vitro and complex microbiota settings.

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Section: Methodsmentioning

confidence: 99%

PUPpy: a primer design pipeline for substrain-level microbial detection and absolute quantification

Ghezzi,

Fan,

et al. 2024

mSphere

View full text Add to dashboard Cite

show abstract

“…Facility at the University of British Columbia. For samples collected 3 days after cessation of DSS, library preparation targeting the V4 hypervariable region of the 16S rRNA gene was performed at the Gut4Health facility using established protocols (De Wolfe and Wright, 2023). Amplification of the V4 region was performed with 515F/926R primers (515F, 5'-GTGYCAGCMGCCGCGGTAA-3'; 926R, 5'-CCGYCAATTYMTTTRAGTTT-3').…”

Section: Microbiome Sequencing and Analysismentioning

confidence: 99%

Early-Life Gut Inflammation Drives Sex-Dependent Shifts in the Microbiome-Endocrine-Brain Axis

Sullivan,

Sie,

et al. 2024

Preprint

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Despite recent advances in understanding the connection between the gut microbiota and the brain, there remains a wide knowledge gap in how gut inflammation impacts brain development. Microbiota-derived metabolite signaling from the gut to the brain is required for normal development of microglia, the brain's resident immune cells. Disruption of the microbiota-brain communication has been linked to impaired behaviours and Autism Spectrum Disorder. We hypothesized that intestinal inflammation in early life would negatively affect neurodevelopment through dysregulation of microbiota communication to brain microglia. To test this hypothesis, we developed a novel pediatric model of Inflammatory Bowel Disease (IBD). IBD is an incurable condition affecting millions of people worldwide, characterized by chronic intestinal inflammation, and has comorbid symptoms of anxiety, depression and cognitive impairment. Significantly, 25% of IBD patients are diagnosed during childhood, and the effect of chronic inflammation during this critical period of development is largely unknown. We developed a chemical model of pediatric chronic IBD by repeatedly treating juvenile mice with dextran sodium sulfate (DSS) in drinking water. DSS-treated mice displayed increased intestinal inflammation, altered microbiota and changes in circulating metabolites. We also found that alterations in gut microbiota had long-term impacts on female microglia and male sex-specific behaviours and testosterone regulation, consistent with delayed puberty observed in male IBD patients. Our research expands our understanding of microbiota-microglia communication underlying development. The gut-brain axis is an exciting target for personalized medicine as microbiome manipulations could be feasible for early intervention to reverse deficits due to juvenile inflammation.

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Towards a Standardized Operating Procedure for eDNA‐Based Biomonitoring in Coastal Marine Salmon Aquaculture

Oladi,

Wilding,

Wyness

et al. 2024

Environmental DNA

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The marine aquaculture industry and regulators are in the process of implementing environmental DNA (eDNA) metabarcoding of microbial communities for compliance monitoring. This requires standardization of sampling, laboratory, and data analysis protocols. Towards this goal, we in this study completed two further milestones using samples collected from two Scottish salmon farms: (i) We tested the effect of using two different PCR protocols (i.e., different DNA polymerases, master mixes, and annealing temperatures), which are frequently being used in eDNA biomonitoring of aquaculture installations, for the amplification of the taxonomic marker gene (V3‐V4 hypervariable region of the bacterial 16S rRNA gene). (ii) We quantified sampling background noise obtained from eDNA samples and statistically compared results with the sampling bias observed in macrofaunal samples from the same source sediments. We detected differences in bacterial community structures resulting from the performance of different PCR protocols, profoundly influencing the interpretation of biomonitoring results. Furthermore, we found that sampling‐induced errors for eDNA samples were similar to errors for macrofaunal samples collected according to compliance monitoring protocol (~25% variability in both cases). Finally, we showed that within‐grab variances of microbial community structures were in the same order of magnitude (less than 10× difference in all cases) as the one obtained from replicate grabs collected from the same locale (impact category). Based on our findings, we suggest using a consistent PCR protocol for biomonitoring efforts to improve the comparability of results, especially when different service providers are conducting the biomonitoring. We propose a sampling scheme to be considered in eDNA biomonitoring that includes taking three replicate grabs at each locale, with one replicate sample from each grab. This minimizes sampling‐induced errors and makes upcoming eDNA‐based monitoring results comparable with previous compliance monitoring results obtained from macrofaunal data.

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Multi-factorial examination of amplicon sequencing workflows from sample preparation to bioinformatic analysis

Cited by 9 publications

References 77 publications

PUPpy: a primer design pipeline for substrain-level microbial detection and absolute quantification

PUPpy: a primer design pipeline for substrain-level microbial detection and absolute quantification

Early-Life Gut Inflammation Drives Sex-Dependent Shifts in the Microbiome-Endocrine-Brain Axis

Towards a Standardized Operating Procedure for eDNA‐Based Biomonitoring in Coastal Marine Salmon Aquaculture

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