Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencing

PLOS One | https://doi.org/10.1371/journal.pone.0326053 June 20, 2025 21 / 29 non-target taxa would be amplified as PCR products. These tests found that with zero mismatches, the primer pair hits were 96% Nassellaria, 76–88% Spumellaria, and 100% Collodaria, indicating that they would theoretically amplify the target taxa with limited contamination from other groups. Extracted DNA of each specimen was amplified using the primer pair designed for the appropriate radiolarian order, based on initial taxonomic assignments during shipboard collection and inspection. Although Collodaria are phylogenetically posi- tioned within Nassellaria, it is common practice among radiolarian workers to consider them a distinct order-level group due to their unique characteristics among polycystines: colonial life habit, all described species have photosymbionts, sometimes cells contain multiple nuclei, and some taxa lack a silicified skeleton [5]. The Collodaria primer set was initially designed to capture family Collosphaeridae and limit amplification of the other collodarian groups (because Collosphaeridae is the only family in which all described species build skeletons that can be morphologically identified). However, in practice, these prim- ers amplified specimens belonging to all collodarian families. Primer sequences were obtained from Integrated DNA Technol- ogies (IDT) with M13 tails and a 5’ block, as recommended by Pacific Biosciences (PacBio) for multiplexed amplicon library

preparation (PacBio publication# 101-921-300, revision 2, April 2022). M13 barcodes were also obtained from IDT to allow for asymmetrically-barcoded samples (up to 384 combinations) compatible with PacBio SMRTbell adapter ligation. Target amplification (PCR-1) was conducted in 25 μL reactions, which included: 12.5 μL KAPA HiFi HotStart Ready- Mix, 2.5 μL forward primer (3μM concentration), 2.5 μL reverse primer (3μM concentration), up to 7.5 μL DNA sample, and 7.5 μL–DNA sample volume of molecular biology grade water. Whenever possible, 10ng of DNA was input into the PCR reaction per individual specimen. This amount was typically only attained for colonial specimens, however, as most solitary specimens yielded very low DNA concentrations. Nassellaria and Spumellaria specimens yielded an average of 2.8ng and 3.6ng total DNA, respectively, whereas Collodaria specimens (most of which were colonial) yielded 250ng DNA on average. In cases where a specimen’s DNA concentration was < 0.75ng/μL, the maximum volume allowable in the reaction (7.5 μL) was used in PCR-1. Even reactions with ~0.5ng total DNA input often yielded successful PCR results, so low amounts of raw DNA did not appear to severely impact downstream processing. The thermocycler program for PCR-1 proceeded as follows: initial denaturation at 95°C for 5 minutes, then 35 cycles of denaturation at 98°C for 30 seconds, annealing for 45 seconds at 54°C (for Nassellaria and Spumellaria) or 64°C (for Collodaria), extension at 72°C for 90 sec- onds, followed by a final extension at 72°C for 2 minutes.

Following the first round of PCR, samples were purified with PacBio paramagnetic beads using standard clean-up pro- tocols. Purified PCR-1 products were Qubit-quantified. Specimens that lacked a detectable amount of DNA at this stage were re-run; if they still did not amplify after a second PCR-1 attempt, they were removed from any further processing and analysis. Out of 304 specimens, 54 failed PCR amplification and 18 were lost to a microcentrifuge tube malfunction, leaving a total of 232 successfully-amplified specimens. These samples underwent a second round of PCR (PCR-2) for barcode attachment. The ingredients in PCR-2 were: 12.5 μL KAPA HiFi HotStart ReadyMix, 1.25 μL forward M13 bar- code (10μM concentration), 1.25 μL reverse M13 barcode (10μM concentration), up to 10 μL cleaned PCR-1 product, and 10 μL–PCR-1 product of molecular biology grade water. PCR-1 products were diluted as needed so that 10ng of DNA was input into PCR-2. The following thermocycler program was used: initial denaturation at 95°C for 5 minutes, then 2 cycles of denaturation at 98°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 90 seconds, followed by 25 cycles of denaturation at 98°C for 30 seconds, annealing at 66°C for 30 seconds, and extension at 72°C for 90 sec- onds, with a final extension at 72°C for 5 minutes. A second PacBio bead clean-up was performed prior to Qubit quantification. PCR-2 products were run on SYBR-safe stained 1% agarose gels to confirm amplification in the size range of the targeted amplicon (~1450 bp) and cleanup of

debris fragments were successful. Sample concentrations were standardized and then pooled. The pool was brought to the University of California, Irvine Genomics Research and Technology Hub, where SMRTbell library preparation was performed by staff using PacBio’s SMRTbell 3.0 prep kit. The finished library was sequenced on a single PacBio Sequel II SMRT Cell 8M.