We show in this study that different centers performing membrane-based spoligotyping exhibit different inter laboratory reproducibility rates ranging from 51 to 100% and most of them reaching more than 90% of quality as represented by their pm. One center had a specific detection problem of spacer 39. Ignoring the errors associated with this spacer, it reached a pm as high as the other centers. The choice of the sample was initially meant to include problematic specimens as well as sample representative of the work flow, however a separate statistical analysis between the random work flow and hard-to-type samples was not feasible here (low effective size), hence the discrepancies reported here are upper limits and could have been lower if only random selection had occurred.
The reasons why spoligotyping errors occur can be diverse. First, DNA quality associated to the extraction procedure could be critical. In this study, however, the DNA extraction procedure did not influence the performance of the membrane-based spoligotyping. Nevertheless, we have to keep in mind that we only tested CTAB and thermolyzates and other extraction procedures were not tested. Some centers repeatedly introduced genotyping errors at multiple spacers in the same samples. Some samples may have been of inadequate quality for all spacers to be sufficiently amplified. However, this occurred both for centers using CTAB and thermolyzate DNA, confirming that factors other than extraction procedure, such as the amount of extracted DNA, inclusion of culture medium when preparing bacterial suspensions or possible culture contaminations, may have influenced PCR quality and hence modified the spoligotypes of some samples.
Regarding artisanal process of membrane production, an adequate quantity of specific probe concentrations on the membrane could sometimes be defective which would lead to the same spacer being recurrently problematic in the same center. Also, commercial membranes might experience some spacer problems so internal quality control must be done by each lab. Indeed, we have observed that in Center 11, spacer 39 was affecting the results of 42 strains. However, in other centers, such problems were not observed. We advise positive and negative controls to be included at randomly chosen positions to detect such possible membrane production problems; centers producing their own membranes should also check their production adequately before use.
Third, operator-dependent washing problems could occur on any part of the membrane so that any spacer could be wrongly scored for some isolates. Insufficient washing can lead to false positive detection of a spacer. As an example, spacers 14 or 37 were detected, although they proved to be absent. In contrast, excessive washing will level the signals of several probes down. All centers used their membranes between 8 to 14 times which is under the limit advised by membranes' manufacturer (up to 20 times). However, this might still be too high. We advise users to always record the results with positive and negative controls included at randomly chosen positions to detect the membrane fall down in quality, and possible insufficient washes.
Fourth, there can be intrinsic problem due to the CRISPR structure in MTC: some spacers constantly provide weaker signals that make the distinction between positive and negative values more difficult. For instance, DVR next to spacer 15 (DVR26) harbors a deletion of 4 nucleotides at its 5' end as shown in highly diverse strains . Consequently, PCR primers do not hybridize properly around spacer 15 which likely leads to a lower amplification level of this spacer.
The high-throughput spoligotyping method has the advantage of being more sensitive to detect these slight variations, given the 3D nature of the immobilized probes, which provides more surface to attach more PCR products. Moreover, unspecific hybridization is removed more easily with the microbead technology, making the detection of spacer 15 possible in any sample. Contrarily, in our study, spacer 40 was detected several times by membrane-based spoligotyping, but not by the microbead-based technique (false positive on membrane).
Fifth, the hybridization reading from the film, and the transfer of this information to a digital format have to be handled manually in the membrane-based spoligotyping increasing the transcription error risk; the best procedure to reduce this risk is to have the results read in duplicate by two independent readers, to check reading mistakes and to solve potential discrepancies.
Reading errors are suppressed in the high-throughput method due to an in-house designed automatized routine, which already provides a digital file with a suggested and objective interpretation according to predefined thresholds (cut-off).
As the distribution of wrongly typed spacers is random (see additional file 1), low quality reading and transfer of results may be the major problem in the classical spoligotyping. This can be particularly misleading when errors concern spacers that are used to classify strains into genotype level. For instance, spacer 18 has been proposed to be highly informative to identify the MTC "X" clade strains, and spacer 40 to define the T2 subclade [8, 20].