C100 thermal cycler

Four randomly chosen DNA aliquots (1.4% of total tissue) were selected for COI amplification. In addition, 50 ng DNA from each of the 14 DNA aliquots was pooled to create a sample representing 20% of the total tissue. For each of these five samples, two PCR were run using one N5LCO and one N5HCO fusion primer, each uniquely tagged (S1 Fig). The same PCR master mix was used for all reactions to ensure identical PCR conditions for all replicates. The ten PCR replicates were then run simultaneously in a C100 Thermalcycler (BioRad, Hercules, CA, USA).
The COI fragment was amplified in a PCR reaction consisting of 1× PCR buffer (including 2.5 mM Mg²⁺), 0.2 mM dNTPs, 0.5 μM of each primer, 0.025 U/μL of HotMaster Taq (5Prime, Gaithersburg, MD, USA), 50 ng DNA, and HPLC H₂O to a total volume of 50 μL. The PCR program was as follows: 94°C for 180 s, 30 cycles of 94°C for 30 s, 46°C for 30 s, and 65°C for 150 s, and 65°C for 5 min. PCR products were excised from a 1% TAE agarose gel and purified using the MinElute Gel Extraction Kit (Qiagen, Hilden, Germany). Concentrations were measured using the Qubit 2.0 BR Kit and the library for sequencing was prepared by pooling 12.3 ng of all ten amplicons. Then, paired-end sequencing was carried out by GATC Biotech (Constance, Germany) using the MiSeq with 300 bp paired-end sequencing.

The 601-nt fragment of the 5′-end of the cox1 gene sequence from the mtDNA region was amplified by PCR using a generic invertebrate amplification forward primer, LCO1490 [S0867] (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and a previously-designed reverse primer, Cff-R [S0368] (5′-GAA GGG TCA AAG AAT GAT GT-3′) [2 (link), 15 (link)]. Each reaction was conducted at a final volume of 30 µl, containing 15 µl MyTaq™ Red Mix (Bioline, Eveleigh, Australia) and approximately 2 µl of genomic DNA template. The PCR was conducted using C100 Thermal Cycler (BioRad, Gladesville, Australia) with cycling parameters set as following: denaturing at 95 °C for 1 min followed by 35 cycles at 95 °C for 15 s, 55 °C for 15 s and 72 °C for 10 s, followed by a final elongation for 5 min at 72 °C. All reactions were run with a negative control using sterile PCR-grade water [1 (link), 2 (link)]. Resulting amplicons were visualised on a 1% (w/v) agarose gel with GelRed™ Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA) in 1× TBE buffer to verify product size. Samples found to yield an unambiguous single band of expected size were sent for bidirectional sequencing (Macrogen Ltd, Seoul, Korea). Individual sequences were assembled and aligned against a reference sequence previously submitted to GenBank (KF684884) [2 (link)] using CLC Main Workbench 6.9.1 (CLC bio, Qiagen, Aarhus, Denmark).