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Scientific reports2021; 11(1); 11884; doi: 10.1038/s41598-021-91425-7

Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome.

Abstract: Microbial communities are commonly studied by using amplicon sequencing of part of the 16S rRNA gene. Sequencing of the full-length 16S rRNA gene can provide higher taxonomic resolution and accuracy. To obtain even higher taxonomic resolution, with as few false-positives as possible, we assessed a method using long amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline. Taxonomic assignment had > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, generating similar taxonomic composition as shotgun sequencing. The rRNA operon amplicon sequencing of equine fecal samples underestimated compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but unlinked rRNA genes had a limited effect on the overall results. The rRNA operon amplicon sequencing with the A519F + U2428R primer set was able to detect some kind of archaeal genomes such as Methanobacteriales and Methanomicrobiales, whereas full-length 16S rRNA with 27F + 1492R could not. Therefore, we conclude that amplicon sequencing targeting the rRNA operon captures more detailed variations of equine microbiota.
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Publication Date: 2021-06-04 PubMed ID: 34088956PubMed Central: PMC8178347DOI: 10.1038/s41598-021-91425-7Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article focuses on the development and assessment of a method for analyzing the microbiome found in horse guts through amplicon sequencing of the rRNA operon, offering greater detail and accuracy in identifying different types of bacteria.

Objective of the Study

  • The primary objective of the study was to establish an improved method for the analysis of the equine gut microbiome. This was achieved by using amplicon sequencing targeting the rRNA operon combined with a CCMetagen pipeline, a technique that allows for high taxonomic resolution and minimal false-positives in microbial community studies.

Methodology and Findings

  • The researchers employed long amplicon sequencing, which targets the rRNA operon to achieve high taxonomic resolution and reduce false-positives.
  • The method’s taxonomic assignment showed > 90% accuracy at the species level in a mock sample and at the family level in equine fecal samples, proving its effectiveness.
  • The results were similar to those produced by a more traditional method known as shotgun sequencing.
  • The rRNA operon amplicon sequencing did underestimate the compositional percentages of bacterial strains containing unlinked rRNA genes by a fourth to a third, but these genes had limited impact on the overall results.

Conclusion and Implications

  • The A519F + U2428R primer set used in the rRNA operon amplicon sequencing could detect certain types of archaeal genomes such as Methanobacteriales and Methanomicrobiales, which the sequencing of full-length 16S rRNA with 27F + 1492R could not.
  • Consequently, the researchers concluded that the amplicon sequencing of the rRNA operon provides a more detailed variation analysis of the equine microbiota than alternative methods.
  • This research provides a significant contribution to equine medicine and science, as understanding the microbiome is essential for animal health and disease prevention.

Cite This Article

APA
Kinoshita Y, Niwa H, Uchida-Fujii E, Nukada T. (2021). Establishment and assessment of an amplicon sequencing method targeting the 16S-ITS-23S rRNA operon for analysis of the equine gut microbiome. Sci Rep, 11(1), 11884. https://doi.org/10.1038/s41598-021-91425-7

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 11
Issue: 1
Pages: 11884
PII: 11884

Researcher Affiliations

Kinoshita, Yuta
  • Microbiology Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi, 329-0412, Japan. kinoshita@equinst.go.jp.
Niwa, Hidekazu
  • Microbiology Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi, 329-0412, Japan.
Uchida-Fujii, Eri
  • Microbiology Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi, 329-0412, Japan.
Nukada, Toshio
  • Microbiology Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke, Tochigi, 329-0412, Japan.

MeSH Terms

  • Animals
  • Bacteria / genetics
  • DNA, Bacterial / genetics
  • DNA, Intergenic
  • Feces
  • Female
  • Gastrointestinal Microbiome
  • High-Throughput Nucleotide Sequencing / methods
  • Horses
  • Microbiota
  • Phylogeny
  • RNA, Ribosomal, 16S / genetics
  • RNA, Ribosomal, 23S / genetics
  • Sequence Analysis, DNA / instrumentation
  • Sequence Analysis, DNA / methods
  • rRNA Operon

Conflict of Interest Statement

The authors declare no competing interests.

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Citations

This article has been cited 23 times.
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