DNA extraction, PCR amplification and 18S rRNA gene sequencing
DNA was extracted from 0.25 g of each sediment core sample using the
Qiagen DNeasy PowerSoil Pro extraction kit (Qiagen, Germany) according
to the manufacturer’s protocol. DNA extractions were performed in small
batches in a random order to minimise bias, and a negative control was
included in every other batch. The concentration and purity of each DNA
sample was checked on the NanoDrop 8000 spectrophotometer
(Thermo Fisher Scientific, MA,
U.S.). Extracted DNA samples were stored at -20 °C.
The V4-V5 region of the 18S rRNA gene was amplified with universal
forward and reverse eukaryotic primers, NSF563
(5’-CGCGGTAATTCCAGCTCCA-3’) and NSR951 (5’-TTGGYRAATGCTTTCGC-3’) (Mangotet al ., 2012). Each 50 µL PCR mix contained 0.5 µL of 2000 units
mL-1 Q5 High-Fidelity DNA polymerase, 10 µL of 5x
reaction buffer, 10 µL of 5x high GC enhancer (New England Biolabs, UK),
1 µL of a 10 mM dNTP mix (Bioline, UK), 26.3 µL of molecular grade
water, 0.1 µL of each 100 μM forward and reverse primer, and 2 µL of
DNA. Negative controls were included. The PCR program was set to an
initial denaturing temperature of 94 °C for 5 minutes, followed by 30
cycles of 94 °C for 30 seconds, an annealing temperature of 60 °C for 30
seconds, an extension temperature of 72 °C for 30 seconds, and then a
final extension temperature of 72 °C for 10 minutes. Successful PCR
amplification was confirmed with an agarose gel. PCR product was
purified with the Millipore multiscreen PCR filter plate kit according
to the manufacturer’s protocol (Merck Millipore, MA, U.S.), resulting in
purified PCR product eluted in 35 µL of molecular grade water.
Second step PCR was performed using a dual-indexing approach (Kozichet al ., 2013), and 25 µL reactions contained 0.25 µL of Q5 DNA
polymerase, 5 µL of reaction buffer, 5 µL of high GC enhancer, 0.5 µL of
dNTPs, 7.25 µL of molecular grade water, 5 µL of the indexing primers
(Kozich et al ., 2013), and
2 µL of purified PCR product from the first PCR step. The second step
PCR program was set to an initial denaturing temperature of 95 °C for 2
minutes, followed by 8 cycles of 95 °C for 15 seconds, an annealing
temperature of 50 °C for 30 seconds, an extension temperature of 72 °C
for 30 seconds, and then a final extension temperature of 72 °C for 10
minutes. Successful PCR amplification from the second PCR step was
confirmed with an agarose gel.
PCR product from the second PCR
step was normalised using the Invitrogen SequalPrep normalisation kit
according to the manufacturer’s protocol
(Thermo Fisher Scientific, MA,
U.S.), resulting in 1-2 ng µL-1 of DNA per sample.
Samples were pooled, gel-extracted using the Qiagen MinElute gel
extraction kit according to the manufacturer’s protocol, and the
purified DNA concentration was quantified using the Invitrogen Qubit
dsDNA high sensitivity assay kit with the Qubit 3.0 fluorometer. The
amplicon library was denatured with NaOH, neutralised with HCl, combined
with 10% denatured PhiX, and then diluted with HT1 buffer
(Illumina, CA, U.S.). The library
was heat denatured at 96 °C for 2 minutes and immediately transferred to
an ice bath prior to sequencing on the Illumina MiSeq Platform with a
500-cycle v2 MiSeq reagent kit.