Amplicons that showed polymorphic positions with respect to the BRS sequence, or with critical phylogenetic markers, were replicated at Uppsala University (Sweden) and at the Innovation and Research Centre (San Michele all'Adige, Trento, Italy).
This region was discovered using unions of one, two, or three markers but was replicated only in the single marker analysis (Table 1).
Besides the markers close to the APOE locus, a marker (rs12989701) upstream of BIN1 locus was replicated and the combined analysis reached genome-wide significance level (p = 5E-08).
Each individual was represented by two copies of each sequence to account for both alleles at a heterozygote site in the nuclear markers, while the mitochondrial (ND2) gene was replicated twice to correspond with the two copies of each nuclear gene sequence for the analyses.
Each evolved line had three replicates, while the ancestor was replicated six-fold (three each for the Ara- and Ara+ marker variants).
This was replicated in a third experiment, in which pupil size was measured as a physiological marker of alertness.
Markers that are replicated in the latter part of the replication cycle are located toward a pole prior to replication, with loci replicated by separate forks being at opposite poles and therefore outside of the average replisome position.
However, very few markers have been replicated consistently between studies.
A marker was 'replicated' when it was identified to be statistically significant in Study 1 followed by replication in Study 2. For replicated markers, a combined analysis using data from both studies was carried out to evaluate the overall effect.
In our view, these findings are remarkable for two reasons: first, they re-emphasise the diagnostic potential of individual marker genes, which have now been replicated in an independent thyroid tumour series.
