DNA extraction and PCR amplification
Once in the laboratory, DNA extraction was carried out using the protocol proposed by Corell and Rodríguez-Ezpeleta (2013) with modified durations and incubation step temperatures. Briefly, specimens were initially rinsed with an abundant amount of sterile water in order to remove ethanol and other inorganic compounds. Approximately 650 mg of the washed sample was transferred to 1.5-ml sterile microcentrifuge tubes and centrifuged at 3,500 x g for 10 min to physically separate the biological material from the residual alcohol supernatant. The DNA was extracted using the sodium dodecyl sulfate (SDS) chloroform method, which involved the following sequential steps. (i ) The biological tissues were macerated in 1 ml of SDS buffer [Tris-HCl (10 mM), EDTA (100 mM, pH 8.0), NaCl (200 mM) and SDS (1 %)] until sample homogenization. (ii ) The macerated samples were digested with proteinase K at 20 mg/ml (Sigma Aldrich, St. Louis, USA) for 4 h at 65 ºC. (iii ) The digested samples were centrifuged at 7,500 x g for 15 min, and the supernatant was decanted into new 2-ml sterile microcentrifuge tubes containing phenol:chloroform:isoamyl alcohol (25:24:1), followed by gentle mixing and centrifugation at 12,000 x g for 15 min. This washing step was repeated twice. (iv ) The remaining phenol traces were removed by mixing the sample with chloroform:isoamyl alcohol (24:1) and centrifuging at 12,000 x g for 10 min before transferring the DNA supernatant to new 2-ml sterile microcentrifuge tubes. (v ) DNA precipitation was achieved by adding 95% ethanol and 3 M sodium acetate and incubating the samples at -80 °C for 1 h. (vi ) After centrifugation at 12,000 x g for 20 min, the remaining salts were removed with 200 µL of 80% ethanol. This step was repeated twice. (vii ) Ethanol was removed by decanting, and the DNA pellet was dried at room temperature for ~10 min. (viii ) Purified DNA was re-suspended in 100 µl of Milli-Q water and stored at - 20 ºC until further analysis.
DNA quantity and purity were evaluated with a Qubit 3.0 fluorimeter using a Qubit dsDNA Broad Range assay (Life Technologies, Grand Island, USA) and a Nanodrop 2000 spectrophotometer (Thermo Scientific, Waltham, USA), respectively. Finally, DNA integrity was assessed by electrophoresis and visualized in a 1% agarose gel. To sequence the extracted DNA with Illumina MiSeq technology, we implemented a dual PCR amplification method. During the first PCR round, we amplified the hypervariable V9 region (~130 bp) of small ribosomal 18S and the Leray_Folmer 1 region (~313 bp) of the COI gene. These fragments were amplified with the use of the universal eukaryote primers 1389F (5’-TTGTACACACCGCCC-3’) and 1510R (5’- CCTTCYGCAGGTTCACCTAC-3’; Amaral-Zettler et al., 2009) and mlCOIintF (5’-GGWACWGGWTGAACWGTWTAYCCYCC-3’) and jgHCO2198 (5’-TAIACYTCIGGRTGICCRAARAAYCA-3’; Leray et al., 2013) for 18S rRNA and COI, respectively.
18S rRNA was amplified with KAPA HiFi HotStart DNA Polymerase (KAPA Biosystems, Basel, Switzerland). Each reaction mix contained 1× KAPA HiFi Buffer GC, 0.3 mM of KAPA dNTP Mix, 0.20 µM of each primer, 1 U of HiFi HotStart DNA Polymerase (KAPA Biosystems), and 150 ng of purified DNA in a total reaction volume of 23 uL. Thermal cycling consisted of an initial incubation at 95 °C for 3 min, followed by 25 cycles of 10 s at 98 °C, 30 s at 68 °C, and 30 s at 72 °C, with a final extension of 10 min at 72 °C. The PCR products were visualized in a 2% agarose gel and then purified with Agencourt AMPure XP paramagnetic beads (Beckman Coulter, San Diego, USA).
The COI marker was amplified with a Platinum Taq DNA Polymerase (High Fidelity; Invitrogen, Carlsbad, USA) in a reaction mix containing 1x High Fidelity Buffer, 3.0 mM of MgSO4, 0.2 mM of dNTP Mix, 0.20 µM of each primer, 1 U of Platinum Taq, and 20 ng of purified DNA in a total reaction volume of 20 uL. Thermal cycling consisted of an initial incubation at 94 °C for 3 min, followed by 38 cycles of 30 s at 94 °C, 30 s at 46 °C, and 90 s at 72 °C, with a final extension of 5 min at 72 °C. The PCR products were visualized in a 2% agarose gel and then purified with Agencourt AMPure XP paramagnetic beads (Beckman Coulter).
In the second PCR, we incorporated the Illumina index using the Nextera XT kit following the instructions of the manufacturer using a high fidelity polymerase (KAPA HiFi HotStart ready mix, KAPA Biosystems). The indexing amplification program included 3 min at 95 °C, followed by 12 and 8 cycles (for the 18S and COI primers, respectively) of 30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C, and a final extension of 5 min at 72 °C. The PCR products were visualized in a 2% agarose gel before being cleaned with two rounds of AMPure XP magnetic beads (Beckman Coulter). The first and second purification rounds targeted nonspecific product sizes and the sizes of expected amplicons, respectively. The cleaned PCR results were visualized in 2% agarose gels and quantified with a Qubit 3.0 fluorometer using a Qubit dsDNA High Sensitivity assay (Life Technologies) before normalization to 40 nM. Finally, each library was consecutively diluted until reaching 4 pM and sequenced with 30% PhiX (Illumina) in an Illumina MiSeq machine at 2 x 150 bp and 2 x 300 bp configurations for the 18S and COI markers, respectively.