Comparative study of the validity of three regions of the 18S‐rRNA gene for massively parallel sequencing‐based monitoring of the planktonic eukaryote community

Abstract

The nuclear 18S-rRNA gene has been used as a metabarcoding marker in massively parallel sequencing (MPS)-based environmental surveys for plankton biodiversity research. However, different hypervariable regions have been used in different studies, and their utility has been debated among researchers. In this study, detailed investigations into 18S-rRNA were carried out; we investigated the effective number of sequences deposited in international nucleotide sequence databases (INSDs), the amplification bias, and the amplicon sequence variability among the three variable regions, V1-3, V4-5 and V7-9, using in silico polymerase chain reaction (PCR) amplification based on INSDs. We also examined the primer universality and the taxonomic identification power, using MPS-based environmental surveys in the Sea of Okhotsk, to determine which region is more useful for MPS-based monitoring. The primer universality was not significantly different among the three regions, but the number of sequences deposited in INSDs was markedly larger for the V4-5 region than for the other two regions. The sequence variability was significantly different, with the highest variability in the V1-3 region, followed by the V7-9 region, and the lowest variability in the V4-5 region. The results of the MPS-based environmental surveys showed significantly higher identification power in the V1-3 and V7-9 regions than in the V4-5 region, but no significant difference was detected between the V1-3 and V7-9 regions. We therefore conclude that the V1-3 region will be the most suitable for future MPS-based monitoring of natural eukaryote communities, as the number of sequences deposited in INSDs increases.

Under the canopy: Community-wide effects of invasive algae in Marine Protected Areas revealed by metabarcoding

“…18S in general is known to underestimate the diversity present at the species level in metabarcoding studies (in metazoans in particular, Tang et al, 2012;Leray and Knowlton, 2016), and several species or even several genera can share the same sequence for the v7 region amplified (Guardiola et al, 2016). In addition, several fragments of 18S have been commonly used in metabarcoding, each with its own characteristics (Hadziavdic et al, 2014;Tanabe et al, 2016). The fragment of COI sequenced here, on the other hand, is longer and more variable, thus allowing the delimitation of a much higher number of MOTUs.…”

Novel Diversity of Deeply Branching Holomycota and Unicellular Holozoans Revealed by Metabarcoding in Middle Paraná River, Argentina

“…Both V4 and V9 are considered suitable regions for biodiversity studies given their great variability and the combination of high and low entropy in these sections, which allows forgood primer binding (Behnke et al, 2011;Hadziavdic et al, 2014;Tanabe et al, 2016). They also have been widely used in molecular diversity studies, so the research community knows the advantages and limitations of both regions (AmaralZettler et al, 2009;Stoeck et al, 2010;Massana et al, 2015).…”

An attempt to semi-quantify potentially toxic diatoms of the genus <i>Pseudo-nitzschia</i> in Tokyo Bay, Japan by using massively parallel sequencing technology

“…DNA was extracted shortly after filtration. In preparation for DNA extraction, a 5% Chelex ® suspension (Chelex 100 Molecular Biology Grade Resin; Bio-Rad Laboratories Inc., Richmond, CA, USA) was prepared by dispersing the resin into ultra-pure water (Tanabe et al 2015, Nagai et al 2016a, Nagai et al 2016b. To ensure effective extraction of DNA from planktonic elements trapped on the filters, filters were cut in half and were placed in 1.5-mL tubes (A.150; Assist; Tokyo, Japan) with 150 µL 5% Chelex buffer.…”

“…New sequencing technologies, such as Roche 454 pyrosequencing and the Illumina MiSeq platform, have made it possible to obtain millions of sequence reads in a single experiment, and massively parallel sequencing (MPS) is currently revolutionizing surveys of eukaryotic diversity (Amaral-Zettler et al 2009, Medinger et al 2010, Tanabe et al 2015. Massively parallel sequencing technology is able to detect most eukaryotes present in water samples from various ecosystems, and to detect lowabundance populations within complex eukaryote communities (Cheung et al 2010, Lindeque et al 2013).…”

“…However, MPS techniques remain problematic for two key reasons. First, such techniques are prone to overestimating the number of OTUs (operational taxonomic units), due to the formation of PCR-generated artifacts such as chimeric sequences (Qiu et al 2001) and sequence errors derived from MPS processes (Kunin et al 2010, Schloss et al 2011, Tanabe et al 2015; second, MPS techniques lack taxonomic identification power, due to sequencing length limitations and the limited quantities of sequences available in international nucleotide sequence databases (INSDs) (Bazinet & Cummings 2012, Tanabe & Toju 2013, Tanabe et al 2015. Currently, in MPS surveys targeting the 18S-rRNA gene, the identification of eukaryote species is possible for 15-45% of OTUs detected in 500-1,000 mL seawater (Nagai et al unpublished data).…”