Short COI markers for freshwater macroinvertebrate metabarcoding
- Published
- Accepted
- 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
- 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.
Author Comment
Revision one of the manuscript, incorporating reviewers feedback
Supplemental Information
Suppl. material 1 - Table S1
Primers evaluated in this study
Suppl. material 2: Figure S1
Developed fusion primers for fwh1 and fwh2 on the Illumina high throughput sequencing platform.
Suppl. material 3: Figure S2
Overview of similarity of used inline tags for the fwh1 and fwh2 fusion primers.
Suppl. material 4: Table S2
Overview of the three Romanian macrozoobenthos sampling sites (Z2, L2, R2).
Suppl. material 5: Figure S3
Overview of the macroinvertebrates composition of the three sample sites in Romania.
Suppl. material 6: Table S3
Overview of used tagging combinations for sample multiplexing for sequencing.
Suppl. material 7: Figure S4
Gradient PCR optimisation for the fwh primer sets.
Suppl. material 8: Script S1
JAMP metabarcoding pipeline (used R commands).
Suppl. material 9: Figure S5
Number of raw sequences obtained for each sample after demultiplexing.
Suppl. material 10: Table S4
OTU table for the 52 taxa mock samples sequenced with the fwh1 and fwh2 primer set.
Suppl. material 11: Figure S6
Proportion of shared reads between the two replicates for DceM amplified with the fwh1 primer set.
Suppl. material 12: Figure S7
Sample composition of Romanian macroinvertebrate samples.