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The same region is often found in a larger group of more remotely related proteins with a reduced stringency.
For this reason, we chose to utilize reduced stringency SNP calls from corona_lite and AtlasSNP2.
Thus, we see no evidence of reduced stringency of selection in the Genlisea + Urticularia clade.
Of the 510 genes, 219 had significant female-biased expression (q < 0.5; a reduced stringency cut-off was used given microarray data tend to have higher variance).
Reduced stringency by decreased hybridisation temperature maximized the overall number and intensity of signals, but this was accompanied with 30% increase of unspecific hybridisation signals.
Clearly, validation of results will be even more critical in cross-species microarray experiments because of questions surrounding cross-hybridisation of related genes under conditions of reduced stringency.
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Reducing stringency to <1e-5 in our analysis revealed numerous spurious matches, often to multiple regions in the Medicago genome (data not shown).
We then reduced the stringency and used the set of genes with fold change (both up- and down-regulated genes) values ≥1.2 and p-value of ≤0.05 in the microarray experiment.
Using a reduced FPC stringency (tol = 5, 1e-06) this BAC was near one end of a contig of 70 clones which is estimated to span 1.97 Mb.
Reciprocally, the chance of false positives was increased in the cross-species use of the cDNA spotted arrays because of reduced hybridization stringency.
Note that the results did not change when we reduced the stringency of the ortholog search by performing only a reciprocal best Blast hit search and omitted the additional filtering steps invoked by InParanoid (Remm et al. 2001).
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