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Developing standards for the microbiome field.

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The National Institute for Biological Standards and Control (NIBSC) developed the first reference reagents for microbiome analysis. These standards revealed significant bias in bioinformatics tools, highlighting the need for standardized methods in microbiome research.

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Area of Science:

  • Microbiology
  • Bioinformatics
  • Genomics

Background:

  • Standardization of microbiome analysis methodologies is critical for the research community.
  • Currently, no accredited or certified reference materials exist for microbiome analysis.
  • The National Institute for Biological Standards and Control (NIBSC) has developed novel reference reagents for microbiome analysis.

Purpose of the Study:

  • To develop and evaluate the first reference reagents for microbiome analysis by next-generation sequencing.
  • To establish a framework for evaluating bioinformatics tool and pipeline bias.
  • To assess the impact of reference reagent composition on benchmarking studies.

Main Methods:

  • Development of NIBSC DNA reference reagents: Gut-Mix-RR and Gut-HiLo-RR.
  • Creation of a four-measure framework for bioinformatics pipeline evaluation.
  • Analysis of shotgun sequencing and 16S rRNA sequencing data using various bioinformatics tools.
  • Benchmarking using commercially available mock communities and NIBSC reference reagents.

Main Results:

  • Bioinformatics tools significantly inflate microbiome health metrics, such as diversity estimates.
  • Biases were observed across all tested tools, with a trade-off between sensitivity and false positives.
  • Reference reagent composition influenced benchmarking results, affecting reported measures.
  • NIBSC reference reagents, composed of gut commensal species, presented challenges for most bioinformatics tools.

Conclusions:

  • Widespread adoption of NIBSC reference reagents can standardize gut microbiome analyses if error levels are agreed upon.
  • A collaborative study is proposed for 2020 to facilitate the adoption of these reference reagents.
  • The field should utilize site-specific reagents of high complexity for robust pipeline benchmarking.