Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol

Abstract

Primary and secondary structural data from the internal transcribed spacer two (ITS2) have been used extensively for diversity studies of many different eukaryotic organisms, including the green algae. Ease of amplification is due, at least in part, to the fact that ITS2 is part of the tandemly-repeated rRNA array. The potential confounding influence of intragenomic variability has yet to be addressed except in a few organisms. Moreover, few of the assessments of intragenomic variation have taken advantage of the deep sequencing capacity of sequence-by-synthesis protocols. We present results from our adaptation of the 16S Metagenomics Sequencing Library Preparation/Illumina protocol for deep sequencing of the ITS2 genes in selected isolates of the green algal genus, Haematococcus. Deep sequencing yielded from just under 20,000 to more than 500,000 merged reads, outpacing results from recent pyrosequencing efforts. Furthermore, a conservative evaluation of these data revealed a range of three to six ITS2 sequence haplotypes (defined as unique sets of nucleotide polymorphisms) across the taxon sampling. The frequency of the dominant haplotype ranged from 0.35 to 0.98. In all but two cases, the haplotype with the greatest frequency corresponded to a sequence obtained by the Sanger method using PCR templates. Our data also show that results from the sequencing-by-synthesis approach are reproducible. In addition to advancing our understanding of ribosomal RNA variation, the results of this investigation will allow us to begin testing hypotheses regarding the maintenance of homogeneity across multi-copy genes.

Sanguina nivaloides and Sanguina aurantia gen. et spp. nov. (Chlorophyta): the taxonomy, phylogeny, biogeography and ecology of two newly recognised algae causing red and orange snow

“…In addition, Alanagreh et al . (2017) reported that the dominant haplotype was usually evident from the Sanger sequencing, similar as in cloning approach.…”

Evaluation of the ribosomal DNA internal transcribed spacer (ITS), specifically ITS1 and ITS2, for the analysis of fungal diversity by deep sequencing

“…In past years, there has not been consistent agreement about the selection of ITS1 or ITS2 in studies of fungal diversity. Nilsson et al [ 27 ], Ihrmark et al [ 28 ] and Alanagre et al [ 29 ] stated that ITS2 was the better choice for 454 pyrosequencing or sequencing with Illumina platforms. However, Wang et al [ 22 ] showed that ITS1 might be a better taxonomic DNA barcode than ITS2 in eukaryotes, based on in silico analyses.…”

Evaluating amplicon high-throughput sequencing data of microalgae living in melting snow: improvements and limitations

“…However, the 18S rDNA marker is not sufficiently variable to distinguish among closely related taxa (Hall et al 2010). In contrast, the internal nuclear rDNA transcribed spacer 2 (ITS2 rDNA) inherits high taxonomic resolution, but also in some cases high intragenomic variation (Thornhill et al 2007;Simon & Weiss 2008;Alanagreh et al 2017), which may affect OTU (Operational Taxonomic Unit) clustering and taxonomic identification. Since the rRNA cassette can vary in copy numbers per organism and the ITS regions are free to independently drift within the same organism, a potential overestimation of OTUs may occur, especially in some fungal species (i.e., one species can split into several OTUs (Lindner & Banik 2011;Lindner et al 2013).…”