Data on optimisation of a multiplex HRM-qPCR assay for native and invasive crayfish as well as the crayfish plague in four river catchments

The data presented here corresponds to the research paper “Simultaneous detection of invasive signal crayfish, endangered white-clawed crayfish and the crayfish plague using environmental DNA”. A crayfish-specific assay was designed and optimised using three real-time PCR supermixes (SYBR™ Green, SsoFast™ EvaGreen® and HOT FIREPol® EvaGreen®). Diagnostic high resolution melt (HRM) data from direct application of assay on both ex-situ eDNA water samples and field samples from four catchments (two in Wales, two in England) is presented in this article, displaying positive HRM profiles for invasive signal crayfish (Pacifastacus leniusculus), native white-clawed crayfish (Austropotamobius pallipes) and crayfish plague causal agent (Aphanomyces astaci).


Type of data
Data in full is provided with this article Related research article Robinson, C.V., Uren Webster, T.M., Cable, J., James, J., Consuegra, S. Simultaneous detection of invasive signal crayfish, endangered whiteclawed crayfish and the crayfish plague using environmental DNA. Biological Conservation 222, 241-252. [1] Value of the data The data shows that melting curve differences between native and invasive crayfish can be used for management purposes by screening eDNA water samples.
The protocol successfully amplifies invasive and native crayfish and can detect their infection status.
The comparison of HRM-qPCR outputs using SYBR™ Green and SsoFast™ Evagreen s suggested that the second qPCR mastermix provided greater sensitivity and reproducibility.
Temporal concentration measurements indicated that eDNA degraded 3 Â in 48 h under controlled conditions.

Data
Data presented in Section 1.1 includes a sequence alignment of Pacifastacus leniusculus and Austropotamobius pallipes 16s mtDNA 83 bp product with binding sites respective forward (ApalPlen16S_F) and reverse (ApalPlen16S_R) primers and nucleotide base differences between the two species ( Fig. 1).
In Section 1.2, data is presented on the average eDNA concentrations of tank water samples collected from tanks containing P. leniusculus at three time points (Fig. 2).
The data presented in Section 1.3 consists of the SYBR™ Green Supermix and SsoFast™ EvaGreen s Supermix qPCR qPCR optimization results of both P. leniusculus and A. pallipes DNA, including the qPCR melt curve graphs (Fig. 3), standard curves with efficiency values (Fig. 4) and raw melt data (Table 1). In addition, Subsection 1.3 includes qPCR melt curve graphs (Fig. 5) and raw melt data ( Table 2) for amplifications of mixed proportions of both P. leniusculus and A. pallipes DNA in the same reaction tube and ex-situ P. leniusculus tank eDNA amplifications ( Fig. 5; Table 3). Data on the qPCR melt curve graphs and raw melt data for HOT FIREPol s EvaGreen s qPCR optimisation with P. leniusculus and the crayfish plague causal agent (Aphanomyces astaci) DNA are presented in Subsection 1.3 in Fig. 6 and Table 4.
In Section 1.4, data represents SsoFast™ EvaGreen s qPCR product melt curve graphs (Fig. 7) and raw melt output (Table 5) from positive eDNA water sample amplifications collected in the Bachowey and Duhonw rivers around crayfish traps containing P. leniusculus. Section 1.5 contains both qPCR melt curve graphs and raw melt information from positive amplifications from the Sgithwen and Bachowey catchments using both SsoFast™ EvaGreen s and HOT FIREPol s EvaGreen s mastermixes (Fig. 8, Table 6).   Data displayed in Section 1.6 includes the SsoFast™ EvaGreen s qPCR product melt curve graphs and raw melt data from positive detections of both P. leniusculus and A. pallipes at the same site in the River Medway and Itchen ( Fig. 9, Table 7). To conclude, Table 8 provides raw melt data on the absence of A. astaci DNA at sites in the River Medway and Itchen where both P. leniusculus and A. pallipes DNA were detected.

Experimental design, materials and methods
Methodologies that produced the data presented in this article are fully detailed in [1]. Below, the qPCR protocol for both SsoFast™ EvaGreen s and HOT FIREPol s EvaGreen s are described to complement data provided here.

Sample collection
Water samples were collected at six locations in the River Wye catchment, seven sites in the River Taff catchment, both in Wales, and at 29 sites in the Itchen and Medway rivers, Southern England as detailed in [1]. An ex-situ experiment was also conducted with P. leniusculus in three 2 L isolated tanks from where water samples were collected 24 and 48 h after removal of the crayfish [1].

qPCR analysis protocol
DNA from the ex-situ eDNA and tissue samples for P. leniusculus and A. pallipes were extracted using Qiagen s DNeasy Blood and Tissue Kit (Qiagen, UK). Crayfish specific primers were designed using Primer3, then tested using Beacon Primer Designer (ver. 2.1, PREMIER Biosoft), and finally checked for cross-amplification using NCBI Primer-BLAST [2] and fresh tissue samples as described in [1].   Water samples were amplified in triplicate using optimised SsoFast™ EvaGreen s supermix assay to assess presence of P. leniusculus and A.pallipes through diagnostic melt peak temperature of resulting qPCR products. Reactions were undertaken in 10 ml volumes using a CFX96 Real-Time PCR detection system (Bio-Rad, UK) consisting of 5 ml SsoFast™ EvaGreen s supermix, 0.25 ml each forward and reverse primer (ApalPlen16S), 3.5 ml ultrapure water and 1 ml DNA. PCR protocol began with 15 min of denaturation at 95°C, followed by 40 cycles of 95°C for 10 s and 61.5°C for 30 s. A melt gradient step was applied to the end of RT-qPCR reactions, ranging from 55°C to 95°C in 0.1°C increments. Once qPCR products were analysed for presence/absence of P. leniusculus and A.pallipes, qPCR amplifications were repeated for positive sites using 2 Â HOT FIREPol s EvaGreen s multiplex mix with 0.4 ml of primer mix (5 mM), 6.6 ml of ultrapure water and 1 ml template DNA. Cycling conditions were as follows: activation at 95°C for 12 min, 40 cycles of 95°C for 15 s, 61.5°C for 20 s and 72°C for 20 s. After the PCR reaction, a melt gradient was applied, which ran from 65°C to 95°C by raising 1°C for 10 s each step. Resulting melt peaks from the multiplex qPCR were then assessed to determine presence/absence of A. astaci in P. leniusculus/A.pallipes positive sites.
The results of the ex situ study indicated that DNA concentration decreased slightly but remained fairly constant across the three time points and was still detectable (melt peak above threshold) at the end of the third time point. DNA quantity was fairly uniform across all tanks, which is to be expected as there was equal biomass of crayfish in each tank, which is known to correlate with the amount of eDNA detected in other aquatic species [3,4].
Our approach is still subject to factors affecting the sensitivity of the eDNA analyses, such as number and type of samples collected, volume of water sampled, types of waterbody sampled and differences in laboratory techniques [5][6][7]. Larger water volumes can increase detectability of eDNA, but there is a tradeoff between volume and number of samples, and we have shown that our method can detect infected crayfish even in small volume samples, while allowing to maximize coverage [8][9][10].