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MP and P10 were found to interact upto the highest level of stringency i.e., –Leu –Trp –His –Ade +X Gal as evident from the appearance of blue colonies on the plate similar to MP –VPg interaction, signifying a strong interaction between the two proteins.
As expected, no reads mapped at the highest level of stringency tested, as the requirement for 100% identity would preclude any matches.
The number of mismatches between the reference and tag is user defined, and when unique genomic mappings are selected, only the mappings at the highest level of stringency are retained.
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Overall, the high level of stringency and the potential to search a large library of broadly dispersed random point mutations are the two most likely reasons why we were able to identify subtle surface residues with important structural and functional properties.
False positives, if any, were sifted out in the next stage with a higher level of stringency where the results of BLAST, best reciprocal BLAST hit and HMM based search were compared and common hits satisfying the cutoff criteria were taken as confirmed hits of Ciona.
Thus, using a relatively high level of stringency, 12.5% of the GS20 454 sequences potentially identify novel maize transcripts.
Because of the high level of reproducibility that was achieved, a relatively high level of stringency (p < 0.0001) was chosen for selection of significant differences.
We speculate that this high level of stringency (requiring the activation of four separate reporter genes) could explain why only a single clone was obtained.
While the quality and quantity of EST data varies greatly for each species, we have found that using a high level of stringency (p = 90 or 95) tends to prevent over assembly and helps distinguish between gene family members.
However, due to its high level of stringency and, possibly, to the structural diversity of LTR retrotransposons in the oil palm genome, this analysis was unable to detect related elements in our case (results not shown).
We used a relatively high level of stringency in this and the following case studies in order to focus on the more prominent changes in expression and to minimize false positive findings in the downstream analyses.
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