High sensitivity of 454 pyrosequencing for detection of rare species in aquatic communities

Abstract

Summary Concerns regarding the rapid loss of endemic biodiversity, and introduction and spread of non‐indigenous species, have focused attention on the need and ability to detect species present in communities at low abundance. However, detection of rare species poses immense technical challenges, especially for morphologically cryptic species, microscopic taxa and those beneath the water surface in aquatic ecosystems. Next‐generation sequencing technology provides a robust tool to assess biodiversity, especially for detection of rare species. Here, we assess the sensitivity of 454 pyrosequencing for detection of rare species using known indicator species spiked into existing complex plankton samples. In addition, we develop universal small subunit ribosomal DNA primers for amplification of a wide range of taxa for detailed description of biodiversity in complex communities. A universality test of newly designed primers for the hypervariable V4 region of the nuclear small subunit ribosomal DNA (V4‐nSSU) using a plankton sample collected from Hamilton Harbor showed that 454 pyrosequencing based on this universal primer pair can recover a wide range of taxa, including animals, plants (algae), fungi, blue‐green algae and protists. A sensitivity test showed that 454 pyrosequencing based on newly designed universal V4‐nSSU primers was extremely sensitive for detection of very rare species. Pyrosequencing was able to recover spiked indicator species with biomass percentage as low as approximately 2·3 × 10−5% when 24 artificially assembled samples were tagged and sequenced in one PicoTiter plate (i.e. sequencing depth of an equivalent of 1/24 PicoTiter plate). In addition, spiked rare species were sometimes recovered as singletons (i.e. Operational Taxonomic Units represented by a single sequence), suggesting that at least some singletons are informative for recovering unique lineages in ‘rare biospheres’. The method established here allows biologists to better investigate the composition of aquatic communities, especially for detection of rare taxa. Despite a small‐scale pyrosequencing effort, we demonstrate the extreme sensitivity of pyrosequencing using rare species spiked into plankton samples. We propose that the method is a powerful tool for detection of rare native and/or alien species.

An evaluation of the applicability of microarrays for monitoring toxic algae in Irish coastal waters

“…Entire genomes can now be sequenced using a new technology called Next Generation Sequencing (NGS) which can generate extensive transciptome data of prokaryotes and eukaryotes in a fast and cost effective way (Logares et al 2013;Zhan et al 2013;Kim et al 2014). There are a number of NGS platforms such as 454 pyrosequencing, illumina, SOLiD, HiSeq and MiSeq, PacBio RS all of which use multiple PCR amplification strategies which has been applied in diversity studies of microbial eukaryotes (Kim et al 2014).…”

“…Toxin results: Multi SPR and ELISA , high-throughput sequencing technologies (Logares et al, 2013;Zhan et al, 2013) and microarrays .…”

Ascidians as models for studying invasion success

“…More conveniently, such a strategy can be used to detect target species by sampling water from the areas of concern without ever detecting living animals (Lodge et al 2012;Thomsen et al 2012;Egan et al 2013). Moreover, these genetic techniques are very powerful at detecting species at low population abundance (Zhan et al 2013) and may be used to powerfully screen for all invasive ascidian species simultaneously as long as technical issues are well addressed (e.g., Zhan et al 2014a, b;Zhan and MacIsaac 2015). In addition, to promote community-based monitoring, ascidian identification cards and posters should be produced and distributed to the general public, aquaculture managers, fish harvesters, processing plant managers, and boaters (Sephton et al 2011).…”

“…In addition to effective policy and management solutions, developing practical antifouling technologies that can be used for commercial shipping, recreational boats, and aquaculture could reduce the scale of both primary and secondary introductions (e.g., Cahil et al 2012). Finally, future development and application of robust detection tools such as microarrays and highthroughput sequencing based on environmental DNA may greatly enhance the power of early detection of new infestations (Lodge et al 2012;Thomsen et al 2012;Egan et al 2013;Zhan et al 2013).…”

Rare biosphere exploration using high-throughput sequencing: research progress and perspectives

“…The detection of an array of rare species represents a major technical challenge in all habitats, but particularly so in communities where species are not typically visible, such as microbes (Pedrós-Alió 2012) and plankton (Zhan et al 2013). Recent technological advances have driven rapid development of many DNA-based methods designed to facilitate biodiversity assessment in complex communities (see reviews by Darling and Mahon 2011;Zimmerman et al 2014).…”