2.2 Primer design, DNA extraction, PCR, and sequencing
Reference nucleotide sequences (Ascothoracida and Cirripedia) of
selected loci were downloaded from NCBI GenBank and aligned with
in-house (see below) or NCBI Facetotecta sequences with MAFFT version
7.450 (–auto –maxiterate 1000; Katoh and Standley 2013). The
alignments were imported, visualized, and used to design primers in
Geneious Prime version 2022.0.2. To enhance specificity and
amplification success, we strived to design longer (>20bp)
primers with GC-contents between 30-60%, a GC-clamp of 2 (3’
terminating in at least one G/C to promote optimal binding), a melting
temperature (Tm) between 55°C-68°C, and no runs of
>4 bases (homopolymeric regions, e.g., TGGGGG). We tested a
total of 28 primer pairs (23 new) and performed 2056 PCR reactions using
these. Tables 1, 2, and S2 provide an overview of the primers, their
sequences, and their amplification success with single specimens.
Our goal was to develop and test the efficacy of fast “filter-free”
DNA-extraction methods that amplify single-specimen y-larvae while
retaining their “exuviae” (here understood as any cuticular remains
originating from an acutal molt of after tissue digestion during DNA
extraction) as vouchers. We therefore tested and compared DNA-extraction
using the GeneReleaser® kit (BioVentures, TN, USA), which has previously
proven to be an efficient protocol for retrieving DNA from zooplankton
(Schizas et al., 1997; Böttger-Schnack and Machida, 2011; Watanabe et
al., 2016; Olesen et al., 2022) and a simplified DNeasy method using a
subset of the reagents in the Blood and Tissue kit (QIAGEN, CA, USA). To
this end, and as a part of a larger phylogenetic and barcoding campaign,
we extracted DNA of a total 421 single y-larva specimens, of which nine
were from the Azores, 326 from Sesoko Island (Japan), 4 from Malaysia,
105 from various locations in Taiwan, and 17 from the White Sea
(Russia).
DNA extracts were kept in a freeze Eppendorf© vial rack during
preparation of the PCR assay. We pipetted 12.2 µL filtered
ddH2O, 5 µL “hot start” HOT FirePol® polymerase master
mix (Solis BioDyne, Tartu, Estonia), 0.4 µL of each oligonucleotide
primer (usually ordered as “desalted”), and 3 µL gDNA template to each
vial. For multiplex PCR reactions, which use two or more primer pairs in
combination, we subtracted the equivalent amount of added primer from
the amount of ddH2O. To increase primer annealing
sensitivity, specificity to complex templates (e.g., >60%
G-C content), and yield we generally used a Touchdown PCR profile under
the following conditions: initial denaturation for 15min at 95°C, then
10 cycles of denaturation at 95°C for 1min, annealing at
Tm+10°C decreasing by 1°C/cycle for 30 seconds,
extention at 72°C for 1kb/60secs, then 30 cycles of denaturation at 95°C
for 1min, annealing at Tm-2°C for 30 seconds, extension
at 72°C for 1kb/60secs, final extension at 72°C for 10min, and finally
20min at 4°C to halt the reaction. Primer details including sequences
and annealing temperatures is listed in Supplementary Table 1.
All PCR reactions were conducted in a DNA Engine Thermal Cycler
(Bio-Rad, Richmond, CA, United States) and PCR products were visualized
in agarose gel with varying concentrations depending on fragment size.
DNA sequencing was performed by Genomics BioSci & Tech Ltd. (New Taipei
City, Taiwan) and Macrogen Europe BV (Amsterdam, The Netherlands).
We sequenced genomic DNA templates of three ribosomal loci
(mitochondrial 16S and nuclear 18S, 28S) and two protein-coding genes
(mitochondrial COX1 and nuclear Histone-3) of 74 specimens and added new
sequences to NCBI GenBank (Tables 1, 2, S2). When we sequenced PCR
amplicons, we defined “successful amplification” as those amplicons
that yielded clear gel electrophoresis bands and clean chromatograms
that aligned with our in-house database. Less than 10% of amplicons
were not sequenced but nonetheless yielded clear bands, and these were
computed as successful amplicons.