Short COI markers for freshwater macroinvertebrate metabarcoding

Ecaterina Edith Vamos; Vasco Elbrecht; Florian Leese

doi:10.7287/peerj.preprints.3037v2

Short COI markers for freshwater macroinvertebrate metabarcoding

Ecaterina Edith Vamos ¹, Vasco Elbrecht ¹, Florian Leese^1,2

1 Aquatic Ecosytem Research, University of Duisburg-Essen, Essen, NRW, Germany

2 University of Duisburg-Essen, Centre for Water and Environmental Research (ZWU) Essen, Essen, NRW, Germany

DOI: 10.7287/peerj.preprints.3037v2

Published: 2017-08-09
Accepted: 2017-08-09

Subject Areas: Biodiversity, Bioinformatics, Molecular Biology
Keywords: metabarcoding, COI primers, ecosystem assessment, degraded DNA, eDNA, macroinvertebrates, in silico

Copyright: © 2017 Vamos et al.
Licence: This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.

Cite this article: Vamos EE, Elbrecht V, Leese F. 2017. Short COI markers for freshwater macroinvertebrate metabarcoding. PeerJ Preprints 5:e3037v2 https://doi.org/10.7287/peerj.preprints.3037v2

Abstract

Species diversity of metazoan bulk samples can be rapidly assessed using cytochrome c oxidase I (COI) metabarcoding. However, in some applications often only degraded DNA is available, e.g. from poorly conserved museum specimens, environmental DNA (eDNA) filtered from water or gut content analyses. Here universal primer sets targeting only a short COI fragment are advantageous, as they often can still amplify short DNA fragments. Using PrimerMiner, we optimised two universal primer sets targeting freshwater macroinvertebrates based on NCBI and BOLD reference sequences. The fwh1 and fwh2 primer sets targeting a 178 and 205 bp region were tested in vitro by sequencing previously used freshwater macroinvertebrate mock communities as well as three monitoring samples from Romanian streams of unknown composition. They were further evaluated in silico for their suitability to amplify other insect groups. The fwh1 primer sets showed the most consistent amplification in silico and in vitro , detecting 92% of the taxa present in the mock communities, and allowing clear differentiation between the three macroinvertebrate communities from the Romanian streams. In silico analysis indicates that the short primers are likely to perform well even for non-freshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2+BR2) revealed that detection efficiency is slightly lower for the new primer set. Nevertheless, the shorter new primer pairs might be useful for studies that have to rely on degraded or poorly conserved DNA and thus be of importance for biomonitoring, conservation biological or molecular ecological studies. Furthermore, our study highlights the need for in silico evaluation of primer sets in order to detect design errors in primers (fwhR2) and find optimal universal primer sets for the target taxa of interest.