Using Next-Generation Sequencing for DNA Barcoding: Capturing Allelic Variation in ITS2

Abstract

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 next-generation 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.

DNA barcoding mosquitoes: advice for potential prospectors

“…These methods have been assessed and compared with traditional Sanger sequencing and found to be both superior and more efficient in terms of labour and cost (Shokralla et al 2014;Shokralla et al 2015;Batovska et al 2017).…”

Marine Invertebrate Larvae Associated with Symbiodinium: A Mutualism from the Start?

“…Based on ribosomal, plastid and mitochondrial phylogenies, Symbiodinium are divided in nine clades (A-I, Figure 1) and many subclades (Rowan and Powers, 1991;LaJeunesse, 2001;Pochon et al, 2014). Species-level diversity is still being assessed, mainly through sequencing of the internal transcribed spacer 2 (ITS2, see LaJeunesse and Trench, 2000), either by denaturing gradient gel electrophoresis (DGGE) based separation of ITS2 genomic copies (LaJeunesse and Trench, 2000;LaJeunesse, 2002;Thornhill et al, 2006) or by next-generation sequencing based elucidation of ITS2 diversity (Arif et al, 2014;Batovska et al, 2016;Hume et al, 2016;Ziegler et al, 2017). As a result, many new taxa and/or strains have been described recently (LaJeunesse et al, 2012;Hume et al, 2015;Lee et al, 2015;Ziegler et al, 2017), with probably more than 100 extant species being present in the genus Symbiodinium (LaJeunesse, 2001).…”

“…This approach holds a dual premise: firstly, intragenomic allelic diversity of the ITS2 marker allows to resolve differences within and between individuals and species previously not possible (Batovska et al, 2016); secondly, in the case of Symbiodinium, deep sequencing of ITS2 diversity may uncover symbiont associations previously not detected (Arif et al, 2014;Boulotte et al, 2016). However, the contribution of low abundant Symbiodinium taxa to host resilience remains to be determined (Lee et al, 2016).…”

Intragenomic internal transcribed spacer 2 variation in a genus of parasitoid wasps (Hymenoptera: Braconidae): implications for accurate species delimitation and phylogenetic analysis

“…Before the advent of next‐generation sequencing (NGS) techniques, studies characterizing intragenomic variants of rDNA used methods such as cloning PCR products to create monotypic products for sequencing (eg Králová‐Hromadová et al ., ). However, with the ability to now sequence every product from a PCR‐amplification using NGS, intragenomic variants can be characterized in much greater detail (Ruiz‐Estévez et al ., ; Batovska et al ., ; Cunning et al ., ). With this unprecedented amount of data comes new challenges, including quality control of raw data and recognizing platform‐specific sequencing errors and errors created during PCR amongst true variants.…”

“…Batovska et al . () clustered Illumina‐generated ITS2 reads of mosquitos at 95% sequence identity, but acknowledged that this may have been too low, in one case masking variants that were of high enough abundance to cause issues in the corresponding Sanger sequencing of the specimens, and that subsequent clustering of the reads at 98% identity produced multiple variants. In microalgae, a clustering identity of 97% within samples was shown to collapse variation more likely to be intragenomic, whilst preserving interspecies diversity (Cunning et al ., ).…”