High resolution melt software v3

All sows and pigs used in the experiments were genotyped for LEPR (rs709596309; C>T; on SSC6) single nucleotide polymorphism. Genomic DNA was isolated from biological samples using a standard protocol. Quantification and purity of DNA was assessed by spectrophotometry with a NanoDrop N-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and the integrity was tested by electrophoresis in agarose gels. The LEPR polymorphism was genotyped by real time qPCR (QuantStudio3, Applied Biosystems, Waltham, MA, USA) with High Resolution Melt analysis. Primers used for genotyping the region containing the target [19 (link)] are: forward 5’-CAGAGGACCTGAATTTTGGAG and reverse 5’-CATAAAAATCAGAAATACCTTCCAG. The PCR reaction was performed in a final volume of 5 μl including 1x Thermo Scientific^™ Luminaris Color HRM qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), 0.4 μM of each primer, and 20 ng of genomic DNA. Thermocycling conditions were 50 °C 2 min, 95 °C 10 min, and 40 cycles of 95 °C 15 sec, 60 °C 1 min, followed by a high-resolution melting curve starting with a denaturation at 95 °C for 15 sec, annealing at 60 °C for 1 min and a slow ramp at 0.015 °C/sec up to 95 °C. High Resolution Melt software v3.1 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) was used for the melting data analysis and the genotyping of the samples.

The following positive and negative controls were used: low positive control (synthetic template of single-stranded DNA containing the SARS-CoV-2 N, RdRp and E genes (GenBank NC_045512.2) at 200 copies/μL), very low positive control (synthetic template of single-stranded DNA containing the SARS-CoV-2 N, RdRp and E (GenBank NC_045512.2) at 20 copies/μL) (S1 Table), and a negative template control, where ultrapure water was loaded at the plate instead cDNA. In addition, ultrapure water was also used instead the clinical specimen as a negative control for RNA extractions, in each extraction batch. QuantStudio Design and Analysis Software v1.5.1 (Applied Biosystems, Waltham (Massachusetts), USA) was used to analyze amplification plots and Ct values. High Resolution Melt Software v.3.2 (Applied Biosystems, Waltham (Massachusetts), USA) was used to differentiate between the high-resolution melting curves and obtain Melting Temperature (Tm) values. This software utilizes an improved clustering algorithm that accurately distinguishes between control and variant genotypes, allowing the user to input the expected number of clusters for increased sensitivity for difficult SNP genotyping.

L cells (4 x 10⁵ cells/well) in complete medium were seeded in 6-well plates and incubated until ~ 90% confluency. Cells were adsorbed with medium alone (mock) or co-infected with three independent dilutions of WT and BC reovirus at an MOI of 10 PFU/cell, then inocula were aspirated and replaced with serum-free medium. After 24 h, cell culture supernatants were collected and enriched for large EV, medium EV, and small EV/free virus fractions by sequential differential centrifugation. Infectious units were then isolated by plaque assay. A total of 24 well-separated plaques per fraction per replicate were picked and amplified in L cell monolayers in 24-well plates for 2 days. RNA was extracted using TRIzol (Invitrogen), reverse transcribed using random hexamers, and genotyped using HRM, as previously described, using primers specific for the L2 segment [54 (link)]. Each sample genotype was called by Applied Biosystems High Resolution Melt Software v3.2 and visually verified by comparison with control reactions containing WT RNA, BC RNA, and mixtures (1:2, 1:1, and 2:1) of WT and BC RNA.

L cells (4 x 10⁵ cells/well) in complete medium were seeded in 6-well plates and incubated until ~ 90% confluency. Cells were adsorbed with medium alone (mock) or co-infected with three independent dilutions of WT and BC reovirus at an MOI of 10 PFU/cell, then inocula were aspirated and replaced with serum-free medium. After 24 h, cell culture supernatants were collected and enriched for large EV, and medium EV, and small EV/free virus fractions by sequential differential centrifugation. Infectious units were then isolated by plaque assay. A total of 24 well-separated plaques per fraction per replicate were picked and amplified in L cell monolayers in 24-well plates for 2 days. RNA was extracted using TRIzol (Invitrogen), reverse transcribed using random hexamers, and genotyped using HRM, as previously described, using primers specific for the L2 segment (54 (link)). Each sample genotype was called by Applied Biosystems High Resolution Melt Software v3.2 and visually verified by comparison with control reactions containing WT RNA, BC RNA, and mixtures (1:2, 1:1, and 2:1) of WT and BC RNA.

HRM reactions were performed on 5 μl of single seed DNA extract, in a total volume of 20 μl, using the MeltDoctor Master mix (Life technologies) on a StepOnePlus instrument (Applied Biosystems) following the manufacturers recommendations. TrnL pseudogene was amplified using the primers trnL-Z1-F and trnL-lg-2R at the final concentration of 0.2 μM, rbcL by the primers rbcL-lg-1-F and rbcL-Z2-R at the final concentration of 0.15 μM (Table 2). PCR conditions were adjusted as follow, an initial denaturation of 10 min at 95°C, 45 cycles of 15 s at 95°C, and 1 min at 60°C, a complete denaturation of 10 s at 95°C, 1 min at 60°C, and a continuous melt rising from 60 to 90°C with 0.3% temperature increment every 15 s.
Each extract was run in duplicate, in the presence of the usual positive, negative and process controls and in the presence of reference materials used for HRM profiles analysis. One reference material is required for each HRM profile. These reference materials were previously prepared by extraction of identified seeds using the described protocol and control of the species by sequencing.
Considering the real-time amplification results, only the samples providing cycle threshold (C_t) values below 35 were considered for HRM results analysis. Analysis of the melting profiles was performed using High Resolution Melt Software v3.0 (Applied Biosystems).

Genotyping was performed using real-time PCR followed by high-resolution melt curve (HRM) analysis on a StepOnePlus™ real-time PCR system (Thermo Fisher Scientific, Waltham, MA USA). Reactions were performed using SNP-specific primers (CD40 rs1883832: forward 5′-GCCTGGTCTCACCTCGC-3′ and reverse 5′-GCCCCAGAGGACGCAC-3′; BAFFR rs7290134: forward 5′-GCTGAATGCTGTGGTCTGTAGTG-3′ and reverse 5′-CATGCACATGCCCTCTTTCTG-3′; LtBR rs10849448 forward 5′-CGGCCAGCTCGCTCCAC-3′ and reverse 5′-GCCTCCAGGGCTCCCA-3′) and MeltDoctor™ HRM Master Mix (Thermo Fisher Scientific, Waltham, MA USA). HRM genotyping data were analyzed using the High-Resolution Melt Software v3.0 (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA USA).