Supplementary Material for: Place of concordance-discordance model in evaluating NGS performance

Introduction: Ideally, evaluating NGS performance requires a gold standard; in its absence, concordance between replicates is often used as substitute standard. However, the appropriateness of the concordance-discordance criterion has been rarely evaluated. This study analyzes the relationship between the probability of discordance and the probability of error under different conditions. Methods: This study used a conditional probability approach under conditional dependence then conditional independence between two sequencing results and compares the probabilities of discordance and error in different theoretical conditions of sensitivity, specificity, and correlation between replicates, then on real results of sequencing genome NA12878. The study examines also covariate effects on discordance and error using generalized additive models with smooth functions. Results: With 99% sensitivity and 99.9% specificity under conditional independence, the probability of error for a positive concordant pair of calls is 0.1%. With additional hypotheses of 0.1% prevalence and 0.9 correlation between replicates, the probability of error for a positive concordant pair is 47.4%. With real data, the estimated sensitivity, specificity, and correlation between tests for variants are around 98.98%, 99.996%, and 93%, respectively, and the error rate for positive concordant calls approximates 2.5%. In covariate effect analyses, the effects’ functional form are close between discordance and error models, though the parts of deviance explained by the covariates differ between discordance and error models. Conclusion: With conditional independence of two sequencing results, the concordance-discordance criterion seems acceptable as substitute standard. However, with high correlation, the criterion becomes questionable because a high percentage of false concordant results appears among concordant results

Supplementary Material for: Relative Strengths and Regulation of Different Promoter-Associated Sequences for Various blaSHV Genes and Their Relationships to β-Lactam Resistance

<p><b><i>Aims:</i></b> This work investigated the relative strengths of different <i>bla</i>_SHV promoter-associated sequences and their regulation function in <i>bla</i>_SHV expression and β-lactam resistance. <b><i>Methods:</i></b> Recombinant plasmids with the promoter-associated sequences (P-W, P-S, P-IS, and P-WPD), <i>tac</i> promoter, and combined fragments of promoter and <i>bla</i>_SHV were separately constructed and transformed into <i>Escherichia coli</i> DH5α. The relative strengths of the promoters indicated by the intensities of green fluorescent protein and the mRNA expression levels of <i>bla</i>_SHV were compared. The minimum inhibitory concentration and extended spectrum β-lactamase phenotypes were evaluated. <b><i>Results:</i></b> The relative strengths were ranked as P-<i>tac</i> > P-WPD > P-IS > P-S > P-W. The mRNA expression and β-lactam resistance levels of the different promoter-associated sequence groups were generally consistent with the strength rank, but the extent of <i>gfp</i> and <i>bla</i>_SHV mRNA levels varied significantly in each group. The β-lactam resistance levels were inconsistent with the strength rank in certain <i>bla</i>_SHV groups. In relation to the different promoter-associated sequences,<i> bla</i>_SHV-ESBLs displayed significantly different change modes of β-lactam resistance compared with <i>bla</i>_{SHV-non-ESBLs}. <b><i>Conclusion:</i></b> The mRNA expression and β-lactam resistance of the <i>bla</i>_SHV showed consistencies and inconsistencies with the strengths of the promoter-associated sequences. The mechanisms accounting for these discrepancies need further investigation.</p