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Related Concept Videos

Modern Molecular Taxonomy01:29

Modern Molecular Taxonomy

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Advancements in molecular biology have revolutionized the identification and characterization of bacteria, with multiple methods leveraging DNA sequencing for enhanced precision. As sequencing technologies improve and costs decline, these approaches are increasingly used in clinical, environmental, and evolutionary studies.Multilocus Sequence Typing (MLST) examines several housekeeping genes, essential chromosomal genes encoding cellular functions, to distinguish strains. Approximately...
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Applications of Molecular Taxonomy01:20

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Molecular taxonomy has revolutionized the understanding and classification of bacteria, providing precise insights into their diversity, evolutionary relationships, and ecological roles. By utilizing molecular techniques such as DNA sequencing and fingerprinting, researchers have made significant strides in various fields related to bacterial studies.Resolving Taxonomic AmbiguitiesMolecular taxonomy has been instrumental in distinguishing closely related bacterial species initially thought to...
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Ribosome Profiling02:24

Ribosome Profiling

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Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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Next-generation Sequencing03:00

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The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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RNA-seq03:21

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Related Experiment Video

Updated: Jul 18, 2025

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
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Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

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Amplicon-Based Microbiome Profiling: From Second- to Third-Generation Sequencing for Higher Taxonomic Resolution.

Elisabetta Notario1, Grazia Visci2, Bruno Fosso1

  • 1Department of Biosciences, Biotechnology and Environment, University of Bari Aldo Moro, 70126 Bari, Italy.

Genes
|August 26, 2023
PubMed
Summary

Third-generation sequencing (TGS) offers superior microbiome profiling by analyzing the full 16S rRNA gene length. This full-length approach provides greater taxonomic resolution compared to traditional next-generation sequencing (NGS) methods.

Keywords:
16S rRNA amplicon-based sequencingmetagenomicsmicrobiomemock analysisnext-generation sequencingthird-generation sequencing

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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

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Area of Science:

  • Microbiology
  • Genomics
  • Bioinformatics

Background:

  • 16S rRNA amplicon sequencing is a common method for microbiome profiling.
  • Next-generation sequencing (NGS) typically uses short reads targeting a few hypervariable regions.
  • Advances in third-generation sequencing (TGS) enable full-length gene sequencing.

Purpose of the Study:

  • To compare the taxonomic characterization performance of different 16S rRNA amplicon sequencing approaches.
  • To evaluate single-region, multiplex (NGS short reads), and full-length (TGS long reads) methods.
  • To determine the optimal strategy for deep microbiome taxonomic characterization.

Main Methods:

  • Comparison of three methods: single-region NGS, multiplex NGS, and full-length TGS.
  • Analysis of benchmark microbiome samples with known taxonomic composition.
  • Assessment of classification performance based on hypervariable regions and gene coverage.

Main Results:

  • Different amplicon-based approaches showed varying classification performance.
  • Full-length sequencing using TGS demonstrated the highest discriminating power, reaching species level.
  • Performance was associated with hypervariable regions and target gene coverage.

Conclusions:

  • Third-generation sequencing (TGS) is recommended for enhanced microbiome taxonomic characterization.
  • The full-length approach offers superior resolution compared to NGS-based methods.
  • Further improvements in experimental protocols and bioinformatics are needed for TGS implementation.