Additional file 8 of Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fish: from genome comparisons to high throughput MALDI-TOF typing
Creators: name: Bridel, Sébastien
name: Bourgeon, Frédéric
name: Marie, Arnaud
name: Saulnier, Denis
name: Pasek, Sophie
name: Nicolas, Pierre
name: Jean-François Bernardet
name: Duchaud, Eric
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 8. Quality control andT. maritimumspecies identification. The full dataset is composed of representatives of 24 Tenacibaculum species including 135 isolates belonging to the species T. maritimum. It encompasses 476 independent acquisitions (one acquisition corresponds to an average spectrum of several technical replicates) corresponding to 5102 spectra including technical and biological replicates. This dataset was divided into two groups: the positive control group (T. maritimum isolates) and the negative control group (the type strains of 23 other Tenacibaculum species). In order to confirm that an isolate belongs to the species T. maritimum, the spectra were scanned to identify the 18 T. maritimum monomorphic biomarkers. However, some of these biomarkers could be absent from a number of T. maritimum strains. Reciprocally, strains that do not belong to the T. maritimum species may possess some T. maritimum monomorphic biomarkers. In order to set up a T. maritimum species identity threshold, a value corresponding to the number of monomorphic biomarkers identified in a single sample was computed. The monomorphic biomarkers frequency plot obtained shows a bimodal distribution (Figure A). All samples with a score above 60% correspond to bona fide T. maritimum isolates while all samples with a score below 25% belong to other Tenacibaculum species. True and false positives correspond to isolates correctly and incorrectly identified as T. maritimum (TP and FP), respectively. On the other hand, true and false negatives correspond to correctly and incorrectly rejected isolates (TN and FN, respectively). Positive isolates correspond to those having an identity value above a defined threshold. Using the full dataset, the number of TP and FP and the number of TN and FN were counted. The accuracy of the tool [i.e., (TP + TN)/(TP + TN + FP + FN)] was then computed by increasing the threshold value from 0% to 100% by a 0.1% step. One could observe than the accuracy varies from 0% to 100% and is maximal (i.e., above 97%) between 25% and 60% of threshold value (Figure B). It is therefore proposed that a 60% threshold value safely identifies isolates as belonging to the T. maritimum species. Using this 60% threshold value, only 10 false negatives (i.e., bona fide T. maritimum isolates discarded) and 0 false positive (i.e., not T. maritimum isolates) were found out of 476 acquisitions. Among the 10 false negatives, 3 resulted from the extraction protocol, 3 from the Direct Deposit with Formic Acid (DDFA) protocol and 4 from the ethanol conservation protocol (see section “Testing alternative sample preparation for MALDI-TOF MS”). One can hypothesize that these rare false negatives correspond to technical problems. Indeed, by performing new spectra acquisitions on the 3 isolates previously analyzed by the extraction protocol, the identification score reached at least 88%, far above the safe threshold value of 60%, demonstrating that the original spectra were likely faulty. Finally, spectra from other Tenacibaculum species all had a score below 23%, far below the confidence threshold.