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Mining environmental high-throughput sequence data sets to identify divergent amplicon clusters for phylogenetic
Anna Gimmler1, Thorsten Stoeck1
1Department of Ecology, University of Kaiserslautern, Erwin-Schrödinger-Straße 14, Kaiserslautern, D-67633, Germany.
Environmental Microbiology Reports
|June 11, 2015
Summary
Environmental high-throughput sequencing (envHTS) reveals protistan diversity. This study combines envHTS with PCR and FISH to identify and visualize microbial eukaryotes, advancing ecological and evolutionary insights.
Area of Science:
- Microbial Ecology
- Molecular Biology
- Bioinformatics
Background:
- Environmental high-throughput sequencing (envHTS) is crucial for exploring protistan diversity and distribution patterns.
- Current applications of envHTS in protistan ecology are largely descriptive.
- Massive amplicon datasets offer potential for deeper ecological and evolutionary insights beyond diversity assessment.
Purpose of the Study:
- To develop and demonstrate a strategy for extracting deeper biological information from envHTS data.
- To move beyond descriptive diversity analysis towards phylogenetic and ecological characterization of protists.
- To integrate amplicon sequencing with targeted molecular methods for comprehensive organismal understanding.
Main Methods:
- Utilized a pyrosequenced V4-SSU rDNA dataset from a solar saltern pond.
- Combined Swarm-based blastn network analysis of 11,579 ciliate V4 amplicons to identify divergent clusters.
- Employed targeted PCR for full-length small subunit ribosomal DNA retrieval.
- Designed Fluorescence In Situ Hybridization (FISH) probes for visualization and screening.
Main Results:
- Successfully identified divergent amplicon clusters within the ciliate V4 dataset.
- Enabled phylogenetic placement and improved resolution for specific protistan groups.
- Provided a method to assess the similarity of environmental amplicons to known species.
- Facilitated visualization of the morphotype associated with specific amplicon clusters.
- Demonstrated rapid FISH screening for geographic distribution and abundance assessment.
- Showed utility in monitoring enrichment cultures for protist isolation.
Conclusions:
- The integrated strategy significantly enhances the utility of envHTS data in protistan ecology.
- This approach allows for robust phylogenetic and ecological characterization of environmental protists.
- It bridges the gap between massive sequencing data and detailed organismal biology, including visualization and distribution patterns.
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