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A paired t test (P = 0.05) was used to determine that the duplicate sample sets did not differ significantly.
Specific activity was calculated for each sample by subtracting the value obtained for the sample containing zVAD-fmk from the mean of the duplicate sample.
However the distribution of any probe found to be more than 2-fold up- or down-regulated due to the batch effect in at least 6 of the duplicate sample pairs was significantly biased towards a lower GC-fraction.
However, the variants that were identified in only one of the duplicates were actually also detectable in the duplicate sample, but just below one of the four variant filtering parameters.
Using the 2009 panel one of the duplicates for animal 19, a very low shedder, was misclassified as negative by qPCR when solid culture had returned a result of 2 CFU/tube although the duplicate sample from this animal was correctly identified.
Regarding the prevalence of KIT mutations in PMBL, we had to take the duplicate sample Med-B1 and parental tumor (PMBL.1) into account; we found a total of five mutations in 32 tested samples and determined a mutation frequency of ~15.6% in our cohort.
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Mendelian-inheritance errors were identified by PedManager software, and concordance was determined for the duplicate samples.
Coefficients of variation of <12.0% were observed for positive and negative controls included on 193 plates over a 10-month period and there was a high level of intraassay repeatability with 12.0% of the duplicate samples having CV of >15.0%.
PCR samples were called inconclusive if only one of the duplicate samples tested positive or weakly positive.
Each well was numbered, in duplicate, using a quasi-Latin square that enabled the duplicate samples to be placed randomly on the plate.
The average consensus rate in the duplicate samples (n = 256) was 99.7%, and all the SNPs were in accordance with Hardy-Weinberg equilibrium (all P≥0.01, Table S1).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com