Data on High Resolution Melting (HRM) and phylogenetic analysis of P. ovale wallikeri and P. ovale curtisi

High Resolution Melting (HRM) analysis is a post-PCR analysis method used for identifying genetic variation in nucleic acid sequences. These data are presenting the identity of the 33 samples used for a qPCR-HRM and a nested snapback methods validation. In addition we are presenting the high resolution melting profiles of P. ovale curtisi (Poc) and P. ovale wallikeri (Pow) in the following conditions: after a direct qPCR run and after a nested snapback run. The qPCR-HRM of artificial mixture of Poc and Pow plasmids (200 copies/μl, each) at different proportions are showing the melting pattern of co-infections with both species. The sequencing methodology of the clpc gene fragment of 12 randomly selected samples is described and their likeness to published sequences is shown in a maximum likelihood tree. “Novel high resolution melting and snapback assays for simultaneous detection and differentiation of Plamodium ovale spp.” [1].


Data
This report is presenting in Table 1 the detailed list of all thirty three (33) samples used to validate a qPCR-HRM and a nested snapback methods developed for P. ovale species differentiation [1]. Table 1 is presenting the origin of samples and the results of different genotyping methods (microscopy, nested-PCR,qPCR-HRM and PrimerDesign kit). The P. ovale clpc gene fragments of twelve (12) samples selected randomly were sequenced. The obtained sequences were used for a phylogenetic reconstruction together with previously published P. ovale clpc sequences (Fig. 3). The PCR and HRM melting curves of P. ovale curtisi (Poc) and P. ovale wallikeri (Pow) are shown in Fig. 1 and the differences between the melting temperatures (Tm) are presented in Fig. 2. The nested snapback DTm was 3.74 C and that of the direct qPCR-HRM was 0.2 C. Intermediate Tm values of 71.07 ± 0.05 C (for qPCR-HRM reaction) and 60.0 ± 1.5 C (for nested snapback reaction) were observed with artificial mix of Poc/Pow at the proportions of 8/2, 7/3, 5/5, 3/7 and 2/8 (Fig. 4).

qPCR-HRM and nested snapback assays
The melting curves of Poc and Pow in Fig. 1 and the melting temperatures (Tm) shown in Fig. 2 were obtained by PCR and high-resolution melting reactions using a Roche LightCycler 480 qPCR system Specifications Table   Subject area  Biology  More specific subject area  High resolution melting Assay, Phylogenetic Analysis  Type of data  Table, text file, figure How data was acquired LightCycler 480 qPCR system HRM assays (Roche Diagnostics GmbH) Data format Analyzed Experimental factors Melting program: Denaturation of the PCR product at 95 C for 1 min followed by a cooling to 50 C for 1 min and a continuous heating at 0.02 C/s; Fluorescence acquisition was done from 50 C to 80 C. The clpc fragments and their phylogenetic analysis processes are described in text file. Artificial mix melting profiles of P. ovale curtisi (200 copies/ml) and P.ovale wallikeri (200 copies/ ml) at the following proportion: 8/2; 7/3; 5/5; 3/7; 2/8

Experimental features
The separated melting curves of both P. ovale species are showing the method accuracy and the difference between the melting temperatures. .

Value of the data
The data present the complete list of samples used to validate the qPCR-HRM and snapback assays with their microscopy, nested PCR and PrimerDesign qPCR genotyping data. A phylogenetic tree presents the classification of the Clpc gene sequences of the selected P ovale samples, comparatively to those of other studies. The data show the separated melting curves of the qPCR-HRM assay of the two P. ovale species. Differences between the melting temperatures (Tm) of the qPCR-HRM and the snapback assays are presented in boxplots.
The sequences were edited using Vector NTI version 11.5 and BioEdit software package version 7.2.6. Multiple sequence alignments were performed using the clustal W algorithm, as implemented in MEGA 7, to compare the obtained sequences to a set of published P. ovale clpc sequences from other studies [2e4] retrieved from GenBank (Accession numbers KP050438 e KP050448, AB649417, AY634623, HQ842632, KX611805, LT594596, LT5994519).

Phylogenetic reconstructions
For phylogenetic reconstructions, the most appropriate model of molecular evolution was determined by the Akaike Information Criterion (AIC) using MEGA7. Maximum likelihood (ML) analyses with 1000 bootstrap replicates were performed using the program MEGA7 with the predetermined model of molecular evolution (GTRþIþG for both datasets) using all sites. All the Plasmodium species clustered separately with strong bootstrap support. Additionally, the P. ovale wallikeri and P. ovale  curtisi formed two distinct sub-clusters with strong bootstrap support. Among the 12 samples that were sequenced, 7 (Po1, Po2, Po4; K46, Pro2, Pro6, Pro21) were clustered with P. ovale wallikeri and 5 (Po3, Pro9, Pro12, K28, K41) were clustered with P. ovale curtisi.