Computational protocol: Paralytic Shellfish Toxins and Cyanotoxins in the Mediterranean: New Data from Sardinia and Sicily (Italy)

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[…] The genomic DNA of A. tamarense UNISS5 was extracted from approximately 10–20 mL of culture in logarithmic growth phase. Cells were harvested by centrifugation at 3000 rpm for 15 min. The pellet was transferred to a 2 mL Eppendorf tube and centrifuged again at 10,000 rpm for 5 min. Total genomic DNA was extracted from the resulting pellet using a DNeasy Plant Kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s instructions. The extracted DNA was immediately frozen at −80 °C. A. minutum UNISS3 and UNISS4 were analysed applying the single-cell PCR method []. Individual cells were isolated under an inverted microscope (Zeiss Axiovert 25, Carl Zeiss, Oberkochen, Germany) with a glass pipette and transferred to 200 µL PCR tubes containing 5 µL of lysis solution (0.005% SDS and 400 ng µL−1 Proteinase K). Tubes were briefly centrifuged to ensure that the cells were at the bottom. Tubes were stored at −20 °C. Lysis involved the thawing of tubes and the freezing at −80 °C for at least 10 min, incubation at 60 °C for 30 min and finally at 95 °C for 10 min. The lysates were used immediately, or stored at −80 °C until used for the PCR.The primers D1R and D2C [] were used to amplify the large subunit LSU rRNA gene and the primers ITSF01 and ITS4 [,] were used to amplify the 5.8S rDNA and internal transcribed spacers (ITS1 and ITS2 regions). The PCRs for A. tamarense UNISS5 were carried out in 25 µL reactions containing 1 µL of DNA extract, 0.625 µL of each primer (10 µM), 1.5 µL of dNTPs (200 µM of each), 1.5 µL of MgCl2 (25 mM), 2.5 µL of 10× PCR buffer, and 0.125 µL of Taq DNA polymerase (Qiagen). The PCRs for A. minutum UNISS3 and UNISS4 were carried out in 50 µL reactions containing 1 µL of DNA extract, 4 µL of each primer (10 µM), 1 µL of dNTPs (200 µM of each), 1 µL of MgCl2 (25 mM), 5 µL of 10× PCR buffer and 0.25 µL of Taq DNA polymerase (Qiagen). PCR cycles included one initial step at 95 °C for 5 min followed by 40 cycles at 95 °C for 20 s, 55 °C (for primers D1R and D2C) or 53 °C (for primers ITSF01 and ITS4) for 30 s and at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. Aliquots of the PCR products were electrophoresed in 1.2% agarose gels. The remaining product was stored at 4 °C until sequenced.The sequences obtained for UNISS3, UNISS4 and UNISS5 were aligned with those obtained from GenBank using the MAFFT v.6 program) [] under FFT–NS–i (slow; iterative refinement method). Phylogenetic relationships were done with the maximum-likelihood (ML) method and the GTRGAMMA evolution model on Randomized Axelerated Maximum Likelihood (RAxML) v. 7.0.4 []. Repeated runs on distinct starting trees were carried out to select the tree with the best topology (the one with the greatest likelihood of 1000 alternative trees). Bootstrap ML analysis was done with 1000 pseudo-replicates and the consensus tree was computed with Mr. Bayes []. All analyses were done through the freely available University of Oslo Bioportal []. […]

Pipeline specifications

Software tools MAFFT, RAxML
Application Phylogenetics
Organisms Homo sapiens, Hemisus marmoratus
Chemicals Microcystins