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Related Experiment Video

Updated: Mar 12, 2026

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome
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Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2.

Jana Batovska1, Noel O I Cogan2, Stacey E Lynch2

  • 1BioSciences Research, Agriculture Victoria, AgriBio Centre for AgriBioscience, Bundoora, Victoria 3083, Australia jana.batovska@ecodev.vic.gov.au.

G3 (Bethesda, Md.)
|November 4, 2016
PubMed
Summary

Next-generation sequencing (NGS) offers superior characterization of the Internal Transcribed Spacer 2 (ITS2) marker in mosquitoes compared to Sanger sequencing. This advanced method reveals greater genetic diversity and improves DNA barcoding resolution for mosquito species identification.

Keywords:
CulicidaeNGSamplicon sequencingindelsmicrosatellites

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Area of Science:

  • Molecular Biology
  • Genomics
  • Entomology

Background:

  • The Internal Transcribed Spacer 2 (ITS2) is a widely used DNA barcoding marker.
  • Hypervariability in ITS2 can challenge traditional sequencing methods like Sanger sequencing.
  • Next-generation sequencing (NGS) enables the sequencing of diverse gene variants, including those with SNPs, indels, and microsatellites.

Purpose of the Study:

  • To compare Sanger sequencing and NGS amplicon sequencing for ITS2 characterization in mosquitoes.
  • To assess the suitability of ITS2 as a DNA barcoding marker for mosquito species.
  • To evaluate the allelic diversity of ITS2 within mosquito individuals and species.

Main Methods:

  • Amplicon sequencing of the ITS2 region using both Sanger and NGS platforms.
  • Analysis of 26 mosquito species comprising 88 samples.
  • Comparative assessment of sequencing performance, resolution, and diversity detection.

Main Results:

  • NGS provided more extensive characterization of the ITS2 region than Sanger sequencing.
  • A total of 382 unique ITS2 alleles were identified across 88 mosquito specimens, revealing significant overlooked diversity.
  • ITS2, particularly with NGS, demonstrated high resolution for species discrimination, comparable to Cytochrome Oxidase I (COI), except for the Culex pipiens complex.
  • Sequence variations like indels and microsatellites within ITS2 alleles were found to be species or genus-specific.

Conclusions:

  • NGS is a powerful and cost-effective tool for sequencing hypervariable markers like ITS2, overcoming limitations of traditional methods.
  • ITS2, when analyzed with NGS, is a valuable DNA barcoding marker for mosquito species identification, offering deep insights into genetic diversity.
  • The study highlights the advantages of NGS in revealing complex genetic variations and improving DNA barcoding efficacy in challenging taxa.