Mining environmental high‐throughput sequence data sets to identify divergent amplicon clusters for phylogenetic reconstruction and morphotype visualization

Abstract

Environmental high-throughput sequencing (envHTS) is a very powerful tool, which in protistan ecology is predominantly used for the exploration of diversity and its geographic and local patterns. We here used a pyrosequenced V4-SSU rDNA data set from a solar saltern pond as test case to exploit such massive protistan amplicon data sets beyond this descriptive purpose. Therefore, we combined a Swarm-based blastn network including 11 579 ciliate V4 amplicons to identify divergent amplicon clusters with targeted polymerase chain reaction (PCR) primer design for full-length small subunit of the ribosomal DNA retrieval and probe design for fluorescence in situ hybridization (FISH). This powerful strategy allows to benefit from envHTS data sets to (i) reveal the phylogenetic position of the taxon behind divergent amplicons; (ii) improve phylogenetic resolution and evolutionary history of specific taxon groups; (iii) solidly assess an amplicons (species') degree of similarity to its closest described relative; (iv) visualize the morphotype behind a divergent amplicons cluster; (v) rapidly FISH screen many environmental samples for geographic/habitat distribution and abundances of the respective organism and (vi) to monitor the success of enrichment strategies in live samples for cultivation and isolation of the respective organisms.

DeSignate: detecting signature characters in gene sequence alignments for taxon diagnoses

“…• Further applications: Next to taxon diagnoses, envisaged applications comprise the support of designing group-specific primers to target certain taxa in natural communities for better diversity estimates [18] and the relation of unknown molecular diversity to morphotypes by using FISH probes [19]. The flourishing field of environmental sequencing generates an enormous amount of data.…”

“…Consider the noisy candidate characters at positions 8,19,20, and 23 in Fig. 1 and a k-window of size k = 5.…”

“…1 and a k-window of size k = 5. Due to the k-window, only the combined characters (19,20), (19,23), and (20, 23) are considered. The combined characters (19,23) and (20, 23) are noisy (CT respectively AT occur in both the query and the reference groups); however, (19,20) is asymmetric since its discriminative power is 1, the query rank is 1 (only CA), and the reference rank is 2 (GA and CT).…”

Untitled

“…After the identification of novel variants of OTUs and amplicons, the next step in the discovery of novel diversity is normally the design of specific primers and probes for the targeted recovery of organisms from environmental samples (Edgcomb et al, 2011b;Gimmler and Stoeck, 2015;Hartikainen et al, 2014;Orsi et al, 2012;Seenivasan et al, 2013). However, this process is time-, cost-, and labor-…”

“…The detection of novel diversity, in specific, uses a strategy that detects reads distantly related to previously sequenced species (e.g. Berney et al, 2013;Dunthorn et al, 2014b;Edgcomb et al, 2011b;Filker et al, 2014;Hartikainen et al, 2014;Gimmler and Stoeck 2015). While the detection and description of novel protists is a central task since our understanding of their diversity is far from complete , our ability to detect novel diversity in molecular environmental studies is affected by the way in which we cluster reads into operational taxonomic units (OTUs).…”