DOI: 10.5068/d1j951
title: MiSebastes: An eDNA metabarcoding primer set for rockfishes (genus Sebastes) supplemental materials

description: Environmental DNA (eDNA) is a promising biomonitoring tool for marine
 ecosystems, but its effectiveness for North Pacific coastal fishes is
 limited by the inability of existing barcoding primers to differentiate
 among rockfishes in the genus Sebastes. Comprised of 110 commercially and
 ecologically important species, this recent radiation is exceptionally
 speciose, and exhibits high sequence similarity among species at standard
 barcoding loci. Here, we report new Sebastes-specific metabarcoding
 primers that target mitochondrial cytochrome B. Amongst the 110 Sebastes
 species, 85 unique barcodes (of which 62 are species-specific) were
 identified in our amplicon region based on available reference sequences.
 The majority of the remaining barcodes are shared by only two species.
 Importantly, MiSebastes yield unique barcodes for 28 of 44 commercially
 harvested species in California, a dramatic improvement compared to the
 widely employed MiFish-U 12S primers which only recover one of 44. Tests
 of these primers in an aquarium mesocosm containing 16 rockfish species
 confirms the utility of these new primers for eDNA metabarcoding,
 providing an important biomonitoring tool for these key coastal marine
 fishes.
description: To test the utility of the
 MiSebastes primer set for eDNA metabarcoding, we collected water samples
 from the kelp forest tank at the California Science Center (700 Exposition
 Park Drive, Los Angeles, CA 90037), a 700,000 L, 10 m deep aquarium
 exhibit containing 16 <i>Sebastes</i> species. The water for
 the aquarium is sourced from a coastal site off of Catalina Island and
 transported by barge and truck to the aquarium. The transit time of the
 water is often longer than 24 hours, and the water is UV sterilized
 through the aquarium’s filtration system. We collected triplicate
 biological replicates of 1 L at both the surface and bottom (one meter off
 the bottom of the tank) of the well-mixed aquarium (total = 6 samples). We
 filtered water samples through a 0.2 mm Sterivex (MilliporeSigma, Burlington,
 MA, USA) filter using a gravity filtering method and stored
 filtered eDNA at -20°C for transport back to the lab at UCLA (Miya et al.
 2016). We extracted eDNA from the filters using the Qiagen
 DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) following the
 modifications of Spens et al. (2017), in which ATL buffer and proteinase K
 are added directly to the sterivex filter cartridge. Cartridges were
 incubated overnight and then transferred to 1.7 mL tubes using a 3 mL
 syringe. Equal amounts of sample, AL buffer, and 0 ˚C ethanol were added.
 The protocol then followed the manufacturer’s instructions. We stored
 extracted eDNA at -20ºC until PCR amplification. We amplified
 extracted eDNA with the MiSebastes<i> </i>primers employing
 the same thermocycling parameters, above, but tripled the volume of
 template DNA from 1 mL to 3 mL to improve PCR sensitivity and performance.
 We decided to triple the volume of template DNA following the results of
 our first PCR tests for this primer set using only 1 mL of starting
 template, which resulted in very faint bands after running PCR product
 through electrophoresis on a 2% agarose gel. The PCR reaction volume was
 thus as follows: 3 mL of template DNA, 1 mL of each primer (10 mM) 12.5 mL
 of QIAGEN Multiplex Taq PCR 2x Master Mix, 2.5 mL of Q solution, and 5 mL
 of nuclease-free water. To minimize PCR dropout, we had three technical
 replicates for each sample by conducting all PCR reactions in triplicate
 (Doi et al. 2019), and all PCR reactions included negative and positive
 controls. Following PCR, we confirmed successful amplification and correct
 product size through electrophoresis on a 2% agarose gel. To compare
 MiSebastes to MiFish-U primers (Miya et al. 2015), we repeated the above
 using the<i> </i>MiFish-U<i> </i>primers (Table 1)
 on the three surface samples. The bottom samples were not sequenced with
 the MiFish-U primers. We used a similar touchdown PCR protocol, but the
 touchdown annealing temperatures began with an initial temperature of
 69.5°C for 30 s and subsequently decreased by 1.5°C every cycle until 50°C
 was reached. The reaction volume was as follows: 1 mL of template DNA, 5
 mL of each primer (2 mM), 12.5 mL of QIAGEN Multiplex Taq PCR 2x Master
 Mix, and 1.5 mL of nuclease-free water. Template volume for
 MiFish-U<i> </i>PCR differed from MiSebastes<i>
 </i>so that both yielded PCR products of similar strength.
 We
 prepared PCR products for sequencing following the CaleDNA protocol (Meyer
 et al. 2019). The three technical replicates for each sample were
 sequenced separately for the MiSebastes primer set; the technical
 replicates for each sample were pooled before being sequenced for the
 MiFish-U<i> </i>primer set. The difference in pooling protocol
 is due to the fact that the MiFish-U samples had been prepared for a
 previous project for which the protocol dictated that PCR products be
 pooled prior to sequencing. However, because MiFish-U and
 MiSebastes<i> </i>results were summed across all samples and
 technical replicates, this difference in methodology had a negligible
 effect on comparisons of overall primer taxonomic resolution. Taxonomic
 detections by individual technical replicates for MiSebastes samples are
 shown in Supplementary Figure 1. We generated two
 sequencing libraries (one for the MiFish-U samples and one for the
 MiSebastes samples) through an indexing PCR using IDT for Illumina Nextera
 UD Indexes Sets A and D (Illumina, San Diego, CA, 92122) and KAPA HiFi
 HotStart Ready Mix (Kapa Biosystems, Wilmington, MA, USA). This second
 indexing PCR was performed using a 25 μL reaction mixture containing 12.5
 μL of Kapa HiFi Hotstart Ready mix, 0.625 μL of primer i7, 0.625 μL of
 primer i5, and 10ng of template DNA, and used the following thermocycling
 parameters: denaturation at 95˚C for 5 min, 5 cycles of denaturation at
 98˚C for 20 sec, annealing at 56˚C for 30 sec, extension at 72˚C for 3
 min, followed by a final extension at 72˚C for 5 min. We ran all indexed
 PCR products on a 2% agarose gels to ensure successful PCR and correct
 product size. We then cleaned the pooled samples using Serapure magnetic
 beads (Faircloth and Glenn 2014) and quantified cleaned PCR product
 concentrations using the high sensitivity Quant-iT™ dsDNA Assay Kit
 (Thermofisher Scientific, Waltham, MA, USA) on a Victor3 plate reader
 (Perkin Elmer Waltham, MA, USA). Following quantification, the indexed
 samples were pooled in equimolar concentration so that the final library
 (volume 300 μL) had a DNA concentration of 20 ng/μL. Because the samples
 were sequenced together with samples for other projects, there were 57
 samples in the library that contained the MiFish-U samples and 91 samples
 in the library that contained the MiSebastes samples (Supplementary Table
 4). Lastly, we sequenced the libraries on a MiSeq PE 2x300bp at the
 Technology Center for Genomics & Bioinformatics (University of
 California – Los Angeles, CA, USA), using Reagent Kit V3 with 20% PhiX
 added to all sequencing runs. We processed
 metabarcoding sequences from the MiSebastes primer set using the
 <i>Anacapa Toolkit</i> (Curd et al. 2019). To maximize
 identification of metabarcode sequences, we used <i>CRUX
 </i>to create a <i>Sebastes</i>-specific<i>
 </i>reference database following the standard <i>CRUX
 </i>parameters. As the <i>Anacapa Toolkit </i>assigns
 taxonomy to ASVs with a confidence score using a Bayesian Lowest Common
 Ancestor (BLCA) method, it can only assign one species to a particular
 ASV. As such, we developed an additional script to the <i>Anacapa
 Toolkit</i> to help assign taxonomy to sequences that are shared
 between multiple species for our metabarcode (https://github.com/markusmin/misebastes_annotator). This add-on for the <i>Anacapa Toolkit</i> identifies all species that share an ASV for our barcode and adds this information to the <i>Anacapa </i>taxonomy output file. We employed standard <i>Anacapa Toolkit </i>parameters and a Bayesian cutoff score of 40 to preserve the low-confidence species-level annotations caused by multiple <i>Sebastes </i>species sharing the same barcode for the MiSebastes primer set. Lowering the Bayesian cutoff score from the standard value of 60 to 40 was necessary to allow the MiSebastes-specific annotation script to identify instances where multiple species share the same barcode, as this script uses our <i>in silico</i> PCR results to add additional species that are known to share the same barcode to the taxonomic assignments from the <i>Anacapa Toolkit</i>. We processed metabarcoding sequences from the MiFish-U primer set using the <i>Anacapa Toolkit</i> (Curd et al. 2019) following standard parameters and a Bayesian cutoff score of 60 (Gao et al. 2017). We assigned taxonomy to MiFish-U data using a California Current-specific fish <i>12S</i>-specific reference barcode database (Gold et al. 2021).