Unveiling invasive insect threats to plant biodiversity: Leveraging eDNA metabarcoding and saturated salt trap solutions for biosurveillance

Library preparation The COI gene high-throughput sequencing library was prepared through a two-step PCR pro- cess/following the same Hanner Laboratory standardized protocols and already published [4, 25]. In the first PCR, a ~407 base-pair segment of the mitochondrial genome (mtDNA) was amplified as a single fragment for all samples. The forward primers mLepF1_MiSeq/RonM- WASPdeg_MiSeq and reverse primers LepR1_MiSeq/HCO2198_MiSeq were used, along with Illumina adaptors. Each reaction included a 25 μL final volume with 2.5 μL of DNA template, 12.5 μL of 2X KAPA HiFi HotStart Ready Mix, and 0.2 μM of each primer. The cycling condi- tions consisted of 94˚C (2 min), followed by 5 cycles of 94˚C (40 s), 45˚C (40 s), 72˚C (1 min), then 35 cycles of 94˚C (40 s), 51˚C (40 s), 72˚C (1 min), and a final extension at 72˚C (5 min) [4]. The PCR products were then analyzed on a 1% agarose gel and purified using 0.8-1x NGS magnetic beads (Machery-Nagel) ratio according to the manufacturer’s instructions. For the second PCR, de novo synthesized index primers equivalent to the Nextera XT Index Kit were incorporated into the COI-Illumina adaptor sequence amplicons. This PCR step introduced unique index primer combinations for each sample. Reactions were set up in a final volume of 50 μL, containing 25 μL of 2X KAPA HiFi HotStart Ready Mix, 10 μL of molec- ular biology grade water, 5 μL of each index primer (10 μM), and 5 μL of the first cleaned-up PCR product. The PCR profile included an initial denaturation at 95˚C for 3 minutes, followed

by 8 cycles of 95˚C (30 s), 55˚C (30 s), 72˚C (30 s), and a final extension of 72˚C (5 min) [4, 26]. The PCR products were visualized on a 1% agarose gel, purified with a 0.6-1x NGS magnetic beads (Machery-Nagel) ratio, following the manufacturer’s instructions. The second PCRs were conducted in separate rooms and PCR workstations from the first PCR to minimize contamination. DNA extracts and post-PCR products were treated and pro- cessed in different areas, following standard protocols for eDNA library preparation in the Hanner laboratory. High-Throughput sequencing The Genomics Facility of the Advanced Analysis Centre (AAC) at the University of Guelph conducted the sequencing process. To prepare the sequencing libraries, the SequalPrep Nor- malization Kit from Thermo Fisher Scientific was used for normalization. The libraries were then pooled and quantified with the Qubit dsDNA HS assay kit (Thermo Fisher Scientific), while fragment size was checked using a Bioanalyzer HS DNA Chip from Agilent. Only librar- ies that passed the quality control were selected for sequencing. An Illumina MiSeq System with a MiSeq reagent kit v3 (600 cycles) was employed for sequencing, with each sample lim- ited to 1% of the run (with a maximum of 100 samples per MiSeq run). The MiSeq Reporter software was utilized for demultiplexing and adapter trimming, resulting in two paired-end raw FASTQ files per sample. Data analysis The quality control process for each raw data file (FASTQ files) involved assessing base

sequence quality scores, per base sequence content, sequence length distribution, sequence duplication levels, overrepresented sequences, and adapter content using FastQC (http://www. bioinformatics.babraham.ac.uk/projects/fastqc/). Summary reports for each dataset were gen- erated using MULTIQC [27]. For COI data analysis, mBRAVE (multiplex barcode research and visualization environment) (http://www.mbrave.net/) was utilized. mBRAVE is a multi- user platform that supports the storage, validation, and visualization of High-Throughput Sequencing (HTS) data. The specific parameters set within the mBRAVE platform were the same already established in previous analyses conducted by our laboratory and proven to be PLOS ONE Unveiling invasive insect threats to plant biodiversity PLOS ONE | https://doi.org/10.1371/journal.pone.0290036 August 11, 2023 6 / 24