Short COI markers for freshwater macroinvertebrate metabarcoding
نویسنده
چکیده
Species diversity of metazoan bulk samples can be rapidly assessed using Cytochrome c oxidase I (COI) metabarcoding. However, in cases where only degraded DNA is available, e.g. from poorly conserved museum specimens, eDNA filtered from water or gut content analyses, universal primer sets that amplify only a short COI fragment are advantageous. 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 vivo by sequencing previously used freshwater macroinvertebrate mock communities of known composition and three monitoring samples from Romanian streams. 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 vivo, 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 nonfreshwater insects. Comparing the performance of the new fwh1 primer sets to a highly degenerate primer set targeting a longer fragment (BF2/BR2) revealed that 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.
منابع مشابه
Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment
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