Quantitative sequencing
Simultaneous quantification and sequencing of the extracted eDNA were
performed by quantitative sequencing (qSeq) as previously described
(Hoshino and Hamada, 2017; Hoshino and Inagaki, 2017). First, a single
primer extension (SPE) was performed. The 20 µL SPE reaction mixture
consisted of 1 × PrimeSTAR Max premix (Takara bio), 300 nM
qSeq-MiFish-U-F (Table 1), and 2 µL of the extracted DNA. SPE was
initiated by denaturation at 94°C for 1 min, followed by cooling to 60°C
at 0.3°C/s, incubation at 60°C for 1 min, and final extension at 70°C
for 10 min. Subsequently, the excess primers that caused the
incorporation of random sequence tags were completely digested by adding
4 µL of Exonuclease I (Takara bio, 5 U/µL) to the SPE mixture. The
digestion was performed at 37°C for 120 min, followed by the
inactivation of Exonuclease I at 80°C for 30 min. The first-round PCR
mixture (25 µL) contained 12.5 µL of PrimeSTAR Max premix, 0.3 µM each
of qSeq-MiFish-U-R and F2 primers (Table 1), and 2 µL of the SPE
product. After 40 cycles of amplification were performed at 98°C for 10
s, 55°C for 5 s, and 72°C for 5 s, the amplification product was
subjected to agarose gel electrophoresis, and the band of the expected
size was removed and purified using Nucleospin Gel and a PCR Clean-up
column (Takara Bio). The qSeq-MiFish-U-R primer also contains eight N
bases to increase the complexity for improving the sequencing quality,
and thus, PhiX was not added in this study. Finally, a 2nd-round PCR was
performed to add an index for Illumina sequencing as described elsewhere
(Caporaso et al., 2011). The indexed PCR amplicon was purified using
AMPure XP beads (Beckman Coulter, Indianapolis, IN) followed by
sequencing using a MiSeq platform with MiSeq Reagent Kit v3 for 600
cycles (Illumina).