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.