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Batovska_et_al_2016_G3.pdf (2.53 MB)

Using next-generation sequencing for DNA barcoding: capturing allelic variation in ITS2

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journal contribution
posted on 2021-01-05, 02:30 authored by Jana Batovska, Noel CoganNoel Cogan, Stacey E Lynch, Mark Blacket
© 2017 Batovska et al.

Internal Transcribed Spacer 2 (ITS2) is a popular DNA barcoding marker; however, in some animal species it is hypervariable and therefore difficult to sequence with traditional methods. With nextgeneration sequencing (NGS) it is possible to sequence all gene variants despite the presence of single nucleotide polymorphisms (SNPs), insertions/deletions (indels), homopolymeric regions, and microsatellites. Our aim was to compare the performance of Sanger sequencing and NGS amplicon sequencing in characterizing ITS2 in 26 mosquito species represented by 88 samples. The suitability of ITS2 as a DNA barcoding marker for mosquitoes, and its allelic diversity in individuals and species, was also assessed. Compared to Sanger sequencing, NGS was able to characterize the ITS2 region to a greater extent, with resolution within and between individuals and species that was previously not possible. A total of 382 unique sequences (alleles) were generated from the 88 mosquito specimens, demonstrating the diversity present that has been overlooked by traditional sequencing methods. Multiple indels and microsatellites were present in the ITS2 alleles, which were often specific to species or genera, causing variation in sequence length. As a barcoding marker, ITS2 was able to separate all of the species, apart from members of the Culex pipiens complex, providing the same resolution as the commonly used Cytochrome Oxidase I (COI). The ability to cost-effectively sequence hypervariable markers makes NGS an invaluable tool with many applications in the DNA barcoding field, and provides insights into the limitations of previous studies and techniques.


The authors thank the mosquito monitors for collecting mosquito samples; Karen Brown for morphologically identifying the mosquitoes; Hiroshi Shinozuka for guidance on the amplicon sequencing protocol; John Clancy from Westmead Hospital (Sydney) for providing assistance with confirming the identification of uncommon species; the Department of Economic Development, Jobs, Transport and Resources for project funding; and the Victorian Arbovirus Disease Control Program, which is funded through the Victorian Department of Health and Human Services.

Department of Economic Development, Jobs, Transport and Resources

Victorian Department of Health and Human Services


Publication Date



G3: Genes, Genomes, Genetics






11p. (p. 19-29)


Genetics Society of America



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