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Similarly, a high stringency analysis using DAVID (NIAID, NIH) [ 36] also found these genes to be strongly associated with the immune response and complement activation.
Nemhauser et al. defined marker genes as those genes specifically regulated by one hormone in a high stringency analysis and not by any other hormone even in a lower stringency analysis [ 22].
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A higher stringency analysis was performed by combining the SAM statistical tools with cluster analysis (Eisen et al. 1998) with stringent filter settings.
The high-stringency analysis from this previous work provided a base definition of duplication status for this analysis.
To achieve a high stringency of analysis, we required independent prediction of positive or negative selection by both methods.
We further characterized the assembled contigs by high stringency BLASTX analysis (< 1e-10 confidence, minimum 100 bp high scoring pair (HSP) match length) to protein datasets from three reference sequenced angiosperm genera (Arabidopsis, Populus and Vitis).
Notably, using high stringency in the analysis of the microarray, data showed only twenty one genes were upregulated in common among the three comparative pairwise analyses.
Summarizing, the frequency of annotated isotigs and singletons was significantly higher than previously reported for new generation sequencing de novo transcriptome assemblies of trees like Pinus contorta[ 24], or two oaks species, Quercus petraea and Q. robur[ 30], even though the high stringency of BLASTX analysis.
At higher stringency, our analysis may be more comprehensive.
Only 10 of the rp promoters do not appear to contain conserved binding sites for any of the three factors, and considering the high stringency used for the analysis, the true number may even be lower.
Among these 11 conserved miRNAs, two (mir-9c2 and mir-13b) were missed by our first approach based on homology search because of the high stringency of our blast analysis.
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