Discovery of stable and variable differences in the Mycobacterium avium subsp. paratuberculosis type I, II, and III genomes by pan-genome microarray analysis

Mycobacterium avium subsp. paratuberculosis is an important animal pathogen widely disseminated in the environment that has also been associated with Crohn's disease in humans. Three M. avium subsp. paratuberculosis genomotypes are recognized, but genomic differences have not been fully described. To further investigate these potential differences, a 60-mer oligonucleotide microarray (designated the MAPAC array), based on the combined genomes of M. avium subsp. paratuberculosis (strain K-10) and Mycobacterium avium subsp. hominissuis (strain 104), was designed and validated. By use of a test panel of defined M. avium subsp. paratuberculosis strains, the MAPAC array was able to identify a set of large sequence polymorphisms (LSPs) diagnostic for each of the three major M. avium subsp. paratuberculosis types. M. avium subsp. paratuberculosis type II strains contained a smaller genomic complement than M. avium subsp. paratuberculosis type I and M. avium subsp. paratuberculosis type III genomotypes, which included a set of genomic regions also found in M. avium subsp. hominissuis 104. Specific PCRs for genes within LSPs that differentiated M. avium subsp. paratuberculosis types were devised and shown to accurately screen a panel (n = 78) of M. avium subsp. paratuberculosis strains. Analysis of insertion/deletion region INDEL12 showed deletion events causing a reduction in the complement of mycobacterial cell entry genes in M. avium subsp. paratuberculosis type II strains and significantly altering the coding of a major immunologic protein (MPT64) associated with persistence and granuloma formation. Analysis of MAPAC data also identified signal variations in several genomic regions, termed variable genomic islands (vGIs), suggestive of transient duplication/deletion events. vGIs contained significantly low GC% and were immediately flanked by insertion sequences, integrases, or short inverted repeat sequences. Quantitative PCR demonstrated that variation in vGI signals could be associated with colony growth rate and morphology.

Analysis of residuals in contingency tables: another nail in the coffin of conditional approaches to significance testing.

Omnibus tests of significance in contingency tables use statistics of the chi-square type. When the null is rejected, residual analyses are conducted to identify cells in which observed frequencies differ significantly from expected frequencies. Residual analyses are thus conditioned on a significant omnibus test. Conditional approaches have been shown to substantially alter type I error rates in cases involving t tests conditional on the results of a test of equality of variances, or tests of regression coefficients conditional on the results of tests of heteroscedasticity. We show that residual analyses conditional on a significant omnibus test are also affected by this problem, yielding type I error rates that can be up to 6 times larger than nominal rates, depending on the size of the table and the form of the marginal distributions. We explored several unconditional approaches in search for a method that maintains the nominal type I error rate and found out that a bootstrap correction for multiple testing achieved this goal. The validity of this approach is documented for two-way contingency tables in the contexts of tests of independence, tests of homogeneity, and fitting psychometric functions. Computer code in MATLAB and R to conduct these analyses is provided as Supplementary Material.