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The combination of profiles across the three sub fragments resolved the five different alleles.
Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM.
Since each MLST locus was divided into two or three sub fragments for the HRM analysis, it was necessary to consider the HRM profiles for all of the sub fragments together in order to assign an MLST allele.
Consequently, for each locus two or three sub fragments were analyzed to provide adequate resolution of the known alleles.
For all 47 isolates, successful amplifications were achieved across the 17 sub fragments spanning the seven MLST loci.
However, sub fragments where the alleles differed by only a single SNP often showed readily distinguished melting profiles (e.g., aspA-2, 7 and aspA-1, 8 in Figure 1A and aspA-7 and aspA-1 in Figure 1C).
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Within each sub fragment, the unique sequences had distinct HRM profiles (Figure 1A, 1B, and 1C).
For each sub fragment, the expected 3 to 6 alleles were resolved by HRM as distinct difference plots (Figure 1).
The left fragment (199 bp) contained three SNPs; however, within this sub fragment, there were only three unique sequences – alleles aspA-1 and -8 were identical as were aspA-2 and -7.
For the middle sub fragment the other alleles were particularly difficult to resolve when uncA-17 was present (Figure 1P), but readily distinguished when uncA-17 was excluded (Figure 1Q).
Consequently, the HRM profile for each sub fragment of uncA-17 was highly divergent from the profiles for the other uncA alleles, with appreciably higher values for the relative signal difference (y axis, Figure 1O, P and R).
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