Parafilaroides decorus subsp. repeat, DNA

Williams, Kalani M., Fessler, M. K., Bloomfield, R. A., Sandke, William D., Malekshahi, Clara R., Keroack, Caroline D., Duignan, Pádraig J., Torquato, Samantha D. & Williams, Steven A., 2020, A novel quantitative real-time PCR diagnostic assay for fecal and nasal swab detection of an otariid lungworm, Parafilaroides decorus, International Journal for Parasitology: Parasites and Wildlife 12, pp. 85-92 : 87-88

2.6. Selecting a P. decorus repeat DNA family for qPCR assay design

For each of the 104 repeat families discovered, the number of sequencing reads from P. decorus and each of the three outgroups that matched the repeat family were tallied. The number of reads from each species belonging to each of the repeat families is summarized in Fig. 1 . For the diagnostic assay, a repeat family with zero reads in the three outgroups were selected as candidates. These candidates were then analyzed using BLASTn (http://blast.ncbi.nlm.nih. gov/Blast.cgi) to ensure that the candidate repeat families were not ribosomal or mitochondrial DNA sequences, or a close match to DNA sequences found in other related parasite species, the marine mammal host or human (in case of any contamination during DNA isolation and manipulation). Those repeat families showing any significant matches to other species were eliminated from consideration.

The RepeatExplorer Clustering tool uses the 3D version of the Fruchterman and Reingold algorithm to generate graph layouts of each repeat family, in which individual reads are represented by vertices and similar reads are connected by edges ( Novák et al., 2010). Therefore, clusters composed of very similar reads will form tight clusters in the graph. To quantitatively measure the similarity of reads in a repeat family, the average edge length connecting reads is output for each repeat family. A smaller average edge length denotes greater similarity among reads. To maximize the percentage of the repeat family that would be captured by a single primer and probe set for a diagnostic assay, and therefore increase the sensitivity of the assay, only repeat families forming tight clusters in the graph layout with low average edge lengths were considered for the assay. For the purposes of this paper, a 2D projection of the selected repeat family graph layout is shown ( Fig. 1B ).

After removing those repeat families that had similarity to sequences in other species or did not form tight clusters, the repeat family with the most reads, and therefore the most abundant in the P. decorus genome, was selected for primer and probe design for a quantitative PCR assay. The repeat family selected was the second most abundant in the P. decorus genome of the original candidates. The cluster visualization of this repeat family and its associated statistics are presented in Fig. 1 . For ease of discussion, this cluster was named Pd65 and the repeat family will henceforth be referred to as the Pd65 repeat. The full consensus sequence of the repeat family (GenBank accession no. MT053285) can be found in the supplementary material (Supplementary Sequence 1).

A primer-probe set (based on repeat family Pd 65 identified in Fig. 1 ) for amplifying and detecting P. decorus DNA by qPCR was designed with the PrimerQuest tool offered through Integrated DNA Technologies (Coralville, IA, USA) using standard parameters (http://www. idtdna.com/primerquest/home/ index). The species-specificity of the primer-probe set was assessed using NCBI's Primer Blast tool (http:// www.ncbi.nlm.nih.gov/tools/primer-blast/). The probe was labeled with a 6FAM fluorophore at the 5′ end and was double quenched using the internal quencher ZEN and the 3′ quencher 3IABkFQ (IOWA BLACK). This labeling/quenching combination has been shown to provide superior sensitivity to other probe/quencher systems ( Pilotte et al., 2016) .

The P. decorus repeat family Pd65 ( Fig. 1 ) was amplified using 5′ - GCA GAT AGG AAG AAC CCA CAA- 3′ as the forward primer, 5′ - AGC AAG CTG CTA ACC CTT C - 3′ as the reverse primer, and/56-FAM/AC AGC AGT C /ZEN/A TCG TGT CCA TAC CA /3IABkFQ/ as the probe. The reactions were prepared using Thermo Fisher Scientific's TaqManṜ Universal PCR Master Mix (Waltham, MA, USA) with 200 nM forward and reverse primer concentrations, and 125 nM probe concentration, following the manufacturer's protocols. Cycling conditions were as prescribed by the manufacturer with an annealing and extending temperature of 60 ̊C. One nanogram of input DNA was used for each reaction for a total reaction volume of 7 μL, a volume and concentration that has been proven optimal ( Pilotte et al., 2016). All reactions were run on Thermo Fisher Scientific's StepOne Plus Real-Time PCR system (Waltham, MA, USA). All samples were run in quadruplicate. Mean Cq (quantification cycle) and standard deviation reported in the results were calculated using all four data points from each sample to reduce error.

Figures

Fig. 1. Repeat family selection for the P. decorus diagnostic assay. A. Number of sequencing reads for P. decorus compared to outgroup species reads for each repeat family (1–104) on a log scale. Arrows indicate repeat families with no reads from the outgroup species. Plot was made using Tableau Software, 2019. B. Within a cluster, reads with similar sequences are closer together. Edges connect a read with its closest match (creating a pair) and the length of this edge represents the amount of overlap between the reads. The mean edge width provides context for the lengths in the cluster, so in a cluster with a larger mean edge width the edges are actually longer than edges in a cluster with a smaller mean edge width. Reads therefore may be distant because of sequence divergence, or in the case of a long repeat (more than 150 base pairs), because of a lack of overlap between reads. However, because there will likely be continuous reads covering different regions of the repeat, these longer repeats should still appear as a tight, though possibly larger, cluster. Read dots that stray from the central cluster most likely represent sequence divergence. Higher density therefore indicates lower sequence divergence.