Supplementary Figures
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posted on 2025-03-12, 18:21 authored by Dhanasekaran ShanmugamDhanasekaran Shanmugam, Manali BajpaiManali Bajpai, Ajinkya Khilari- Supplementary Figure 1: Multiplexed nested PCR details. A, schematic depiction of the LSDV genomic coordinates for the target region of the three nest PCRs. B, PCR assays showing the specificity and sensitivity of the multiplexed nested PCR. Lanes 1 - 3, three different negative field samples (a, b & c); Lanes 4 - 6, samples a, b & c spiked with 100 fg of 1 kb template; Lanes 7 - 9, samples a, b & c spiked with 10 fg of 1 kb template; Lanes 10 - 12, samples a, b & c spiked with 1 fg of 1 kb template; Lanes 13 - 15, samples a, b & c spiked with 0.1 fg of 1 kb template; Lanes 16 - 19, four field samples which are positive for LSDV showing the expected 500 bp multiplexed nested PCR product. Lane M - 100 bp DNA size ladder.
- Supplementary Figure 2: LSDV genome amplification and nanopore sequencing using the LSDV_WGSPP7.5kb primer panel. A, Agarose gel electrophoresis confirmation of the ~7.5 kb PCR product obtained from two different field samples (Lane 1 & 2) collected from animals with clinical diagnosis for LSD. Total DNA isolated from skin scab swab samples was used for PCR amplification. Lane M - 1 kb DNA size ladder. B, representative nanopore sequence read count plot for sample PP488405 for which the genome amplification was carried out using the LSDV_WGSPP7.5kb primer panel. The resolution of the plot is at individual base level and red marks indicate the positions at which the read depth was <20X. To obtain good quality genome sequence data that can be used for variant calling, it was necessary to carryout individual PCR amplification for many primer pairs of the panel (see Figure 3 data). Based on observed results the LSDV_WGSPP7.5kb primer panel was not found to be suitable for multiplexed amplification of the LSDV genome from field samples.
- Supplementary Figure 3: Comparative performance of Taq DNA polymerase enzymes for multiplexed PCR amplification of LSDV genome. A, performance of Quantabio Replica hifi tough mix (Lane 1 - 4) and Takara PrimeStar GXL (Lane 5 - 8) enzymes for LSDV genome amplification in multiplexed PCR reactions using the LSDV_WGSPP3.5kb primer panel. Two field samples were used and for each sample pool A and pool B reactions were run (see methods section for details): Lanes 1 & 5 - pool A reaction of sample 1; Lanes 2 & 6 - pool B reaction of sample 1; Lanes 3 & 7 - pool A reaction of sample 2; Lanes 4 & 8 - pool B reaction of sample 2. Lane M - 1 kb DNA size ladder. B, performance of NEB LongAmp (Lane 1 - 6) and NEB Q5 Hotstar High-fidelity (Lane 7 - 12) enzymes for LSDV genome amplification in multiplexed PCR reactions using the LSDV_WGSPP3.5kb primer panel. Three field samples were used and for each sample pool A and pool B reactions were run (see methods section for details): Lanes 1 & 7 - pool A reaction of sample 1; Lanes 2 & 8 - pool B reaction of sample 1; Lanes 3 & 9 - pool A reaction of sample 2; Lanes 4 & 10 - pool B reaction of sample 2. Lanes 5 & 11 - pool A reaction of sample 3; Lanes 6 & 12 - pool B reaction of sample 3. Lane M - 1 kb DNA size ladder.
- Supplementary Figure 4: Co-occurrence of novel mutations in the field strain LSDV genomes. Position-wise list of all novel mutations identified from the LSDV genomes sequenced from 41 field samples analysed in this work is given. Column headers: position - genomic position of the mutation; REF - reference genotype (NC003027.1); ALT - mutant genotype seen in field samples. Mutation colour code: red - non-synonymous; green - synonymous; yellow - indels; white - same as reference.
