Ion torrent one touch system 2

For each PCR, 3 µl of DNA solution was electrophoresed in a 2% agarose gel to check amplification efficiency. SSU rDNA and mini-COI libraries were prepared separately. Next, the amplicons were pooled and purified using a 2% E-Gel SizeSelect II Agarose Gel System (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s protocol. The DNA concentration and fragment length distribution of the libraries were determined using a High-Sensitivity D1000 Screen Tape assay on a 2200 Tape Station system (Agilent Technologies, Inc., Santa Clara, CA, USA). Clonal template amplifications were performed using the Ion Torrent One Touch System II and Ion Torrent OT2 Kit (Life Technologies, Thermo Fisher Scientific) according to the manufacturer’s protocol. For emulsion PCR, SSU rDNA and mini-COI libraries were pooled in a 10:1 ratio. Sequencing was performed using the Ion 540 Kit-OT2 and Ion S5 systems on Ion 540 chips (Life Technologies, Thermo Fisher Scientific), according to the manufacturer’s instructions. Sequencing was designed to yield approximately 10,000 and 1000 reads per SSU and COI amplicons, respectively.

The 18S rDNA and mini-COI libraries were prepared separately. For each PCR reaction, 3 µl was electrophoresed on a 2% agarose gel to check amplification efficiency. Then, the amplicons were pooled and purified using 2% E-Gel SizeSelect II Agarose Gels system (Invitrogen, USA), according to the manufacturer’s protocol. DNA concentration and fragment length distribution of the libraries were established using High Sensitivity D1000 Screen Tape assay on 2200 Tape Station system (Agilent Technologies, USA). For the emulsion PCR, the 18S rDNA and mini-COI libraries were pooled in the 10:1 ratio. Emulsion PCR and sequencing were carried out using the Ion Torrent One Touch System II, the Ion 520 and Ion 530 Kit-OT2, Ion 530 chip and the S5 system (Life Technologies) according to the manufacturer's instructions.