Using DNA metabarcoding to identify a pool of formalin-preserved crustacean larvae
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The research team was interested in using molecular tools to identify to the species level a pool of crustacean larvae which were not possible to identify using morphological methods.
We carried out the DNA isolation from the larval pool following a special protocol for formalin-fixed material. In addition, we extracted DNA from ethanol-preserved specimens that belonged to the four potential crustacean species, to be used as positive controls.
A primer pair was designed by our bioinformatics team to amplify a short-fragment (as recommended when working with degraded DNA) of the COI mitochondrial gene.
In the lab, we verified that by using that primer pair it was possible to amplify DNA from the four positive control samples. Then, we proceeded with DNA metabarcoding library preparation, MiSeq sequencing, and bioinformatic analyses.
For taxonomic assignment, we created a local reference database by combining the COI sequence data available in the DDBJ/ENA/GenBank and BOLD databases for the order Decapoda.
Our results confirmed that the larval pool was composed of one species only (Aristeus antennatus) as all the sequences obtained (even the low-frequency haplotypes) belonged to that species.
This research has been published in the journal Scientia Marina.