Fottea 2019, 19(2):115-131 | DOI: 10.5507/fot.2019.003

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

Stefanie Lutz1*, Lenka Procházková2*, Liane G. Benning1,3,4, Linda Nedbalová2,5, Daniel Remias6
1 GFZ German Research Centre for Geosciences, Telegrafenberg, 14473 Potsdam, Germany; *Corresponding author e-mail: stefanie.lutz@agroscope.admin.ch; current address: Agroscope, Müller-Thurgau-Strasse 29, 8820 Wädenswil, Switzerland
2 Department of Ecology, Faculty of Science, Charles University, Viničná 7, 128 44 Prague 2, Czech Republic; *Corresponding author e-mail: lenkacerven@gmail.com
3 School of Earth & Environment, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK
4 Department of Earth Sciences, Free University of Berlin, 12249 Berlin, Germany
5 The Czech Academy of Sciences, Institute of Botany, Dukelská 135, 379 82 Třeboň, Czech Republic
6 University of Applied Sciences Upper Austria, Stelzhamerstr. 23, 4600 Wels, Austria

Melting snowfields are dominated by closely related green algae. Although microscopy-based classification are evaluable distinction tools, they can be challenging and may not reveal the diversity. High-throughput sequencing (HTS) allows for a comprehensive community evaluation but has been rarely used in such ecosystems. We found that assigning taxonomy to DNA sequences strongly depends on the quality of the reference databases. Furthermore, for an accurate identification, a combination of manual inspection of automated assignments, and oligotyping of the abundant 18S OTUs and ITS2 secondary structure analyses were needed. The use of one marker can be misleading because of low variability (18S) or the scarcity of references (ITS2). Our evaluation reveals that HTS outputs need to be thoroughly checked when the organisms are poorly represented in databases. We recommend an optimized workflow including consistent sampling, a two-molecular marker approach, light microscopy-based guidance, generation of appropriate reference sequences and a final manual verification of taxonomic assignments as a best approach for accurate diversity analyses.

Keywords: 18S rDNA, ITS2 rDNA, high-throughput sequencing, Illumina, oligotyping, OTU clustering, red snow, Sanger, secondary structure, snow algae

Received: October 25, 2018; Accepted: February 6, 2019; Prepublished online: August 1, 2019; Published: October 30, 2019Show citation

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Lutz, S., Procházková, L., Benning, L.G., Nedbalová, L., & Remias, D. (2019). Evaluating amplicon high-throughput sequencing data of microalgae living in melting snow: improvements and limitations. Fottea, 19(2), 115-131. doi: 10.5507/fot.2019.003
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