Library construction and sequencing
We amplified two short overlapping fragments, FC (235 bp) and BR (428 bp), that together form the standard 658 bp DNA barcode region, located at the 5’ end of the cytochrome c oxidase subunit I (COI) gene [23]. These short fragments are expected to enhance PCR success rates and ensure compatibility with read length constraints of the Illumina MiSeq system. The FC and BR fragments were amplified using the primer pairs Ill_LCO1490 and Ill_C_R, and Ill_B_F and Ill_HCO2198, respectively [23]. Forward and reverse primers were tagged with Nextera XT forward or reverse adapters (Illumina, Inc., San Diego, CA, USA), respectively, padded with 0-4 mer nucleotide spacers to increase sequence heterogeneity, as described in [25]. PCRs for both amplicons were carried out using the FastStart Taq DNA Polymerase kit (Roche, Basel, Switzerland), with final concentrations of 1x PCR buffer with MgCl2, 0.2 nM dNTP mix, 1 µg/ml Bovine Serum Albumin (BSA), 250 nM of primer Ill_LCO1490 or Ill_HCO2198 and 750 nM of primer Ill_C_R or Ill_B_F, 1 U Taq polymerase, and PCR grade water up to a total reaction volume of 20 µl. PCR cycle conditions were 95 °C for 5 minutes; 40 cycles of 95 °C for 45 seconds, 50 °C for 45 seconds, and 72 °C for 45 seconds; and 72 °C for 5 minutes (VeritiTM 96-Well Thermal Cycler; Applied Biosystems, Waltham, Massachusetts, USA). Following this, 4 µl of PCR product for each sample was run on a 1.5 % agarose gel to check amplification success. First-step PCR amplicons were cleaned, normalised for maximum concentration, and size selected using SerraMag SpeedBeadsTM magnetic carboxylate modified particles (Cytiva, Marlborough, Massachusetts, US) with 0.8x bead:buffer concentration [26] to remove primer dimers before the second-step PCR.
Second-step PCRs were performed using the KAPA3G Plant PCR kit (KAPABIOSYSTEMS, Cape Town, South Africa), with Fusion primers with custom indices synthesized with P5/P7 Illumina adapters (forward, 5’- AAT GAT ACG GCG ACC ACC GAG ATC TAC AC - [8-mer tag] - TCG TCG GCA GCG TC -3’; reverse, 5’- CAA GCA GAA GAC GGC ATA CGA GAT - [8-mer tag] - GTC TCG TGG GCT CGG -3’)
[27], using 400 nM of each primer and 1.44 μL of first-step PCR product in 18 μL volume. The second-step PCR cycle was 95 °C for 2 min, then five cycles of 95 °C for 20 s, 50 °C for 20 s and 72 °C for 30 s, followed by 72 °C for 2 min (Bio-Rad T100TM Thermal Cycler; Hercules, California, US). Then 2.5 μL of second-step PCR products were run on a 2 % agarose gel to check amplification success.
The amplicons were again cleaned, normalised, and size selected using SerraMag SpeedBeadsTM magnetic carboxylate modified particles (Cytiva, Marlborough, US) with 1.5x bead:buffer concentration. Amplicons were eluted in 15 μL of 0.1x TE buffer, and pooled together at equal volumes to form the amplicon library, which was analysed on the Automated Bioanalysis System LabChip® GX Touch HT (Perkin-Elmer, Waltham, Massachusetts, USA). DNA concentration was estimated by Qubit® 2.0 Fluorometer (Invitrogen, Waltham, Massachusetts, USA), and adjusted to 4 nM. The final library was processed on an Illumina MiSeq 3000 sequencer with 2 x 300 paired-end mode at the Genomics Facility at the University of Auckland, with 10% PhiX spiked in.