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The result is more or less a remake of the great scene in "Sherlock Jr.," where a dozing Buster Keaton dreams himself through a shuffled sequence of backgrounds.
As a negative control we used, in all experiments a shuffled sequence non targeting siRNA from Dharmacon.
The number of possible permutations within this conservative shuffling procedure can be small, and the total amount of compensatory and consistent mutations will be preserved in the shuffled sequence.
We designate negative sites as all possible sites from the shuffled sequence datasets, which can be calculated as 594 × 1870 where 594 is length of the shuffled promoter sequence (600) minus the length of the matrix and 1870 is the number of sequences in the shuffled dataset.
Using real non-orthologous genomic sequence as a control is preferable to using randomized or 'shuffled' sequence as it maintains the complex sequence makeup of true genomic sequences, whereas in shuffled sequences these motifs will be depleted providing a less stringent control.
> -wrap-foot> Boundaries for each alignment were known because the query domain was embedded in shuffled sequence.
Neutral expectations were determined separately for each sequence category, by counting the occurrences of the −10 σ70 hexamers in sets of 1000 shuffled sequences.
Each alignment algorithm was run in triplicate with randomly shuffled sequences for each run, to avoid bias based on sequence input order, and averages of these three runs are reported.
For example, in Figure 1 artificial, randomly shuffled, sequence from the query appears to be homologous to a real protein.
The handful of short peptides that occurred in both the real and shuffled sequence databases were ignored for the purpose of calculating FDR.
The shuffled sequence kept a strict codon usage frequency in the corresponding native sequence.
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