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However, in our bioinformatics analyses, these two binding motifs are not among the most representative sequences in GBRs of glucocorticoid-repressed genes.
By calculating the D m we distinguished the ten most representative sequences and took them as the criteria.
The ten training samples include five most representative sequences of normal sperms and five ones of abnormal sperms.
We removed the redundancy of this alignment (50%) to keep the most representative sequences from the profile.
The general procedure used to define the final DNA barcode is described in the flow chart in Figure 4. Extraction of the N most representative sequences was performed with the Seq_reformat program, a part of the T-COFFEE package [ 37].
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If multiple best scoring hits fulfilled these criteria, the most representative sequence was selected by the following procedure: The average sequence distance (over the length of the whole sequences) between each hit and the other best scoring hits was calculated based on a distance matrix generated in ARB [33].
We considered that the most representative sequence in each group should have the highest number of matching counts with other sequences in the group, and that sequences similar to the representative sequence should belong to that group.
First, the sequences can be sorted — the code selects the most representative sequence, displaying that at the top of the alignment followed by the most similar sequence and so on.
For each cluster, we extracted the most representative sequence (see Materials and Methods), aligned them with MUSCLE (Edgar 2004) and used the resulting multiple sequence alignment to infer a maximum-likelihood tree with RAxML (Stamatakis 2014).
A restrictive 68% confidence interval, corresponding to the mean value plus and minus the standard deviation, was also calculated along with lower and upper intervals to describe the first two properties using the most representative sequence datasets, whereas median, minimum and maximum values were used to report the third characteristic.
Third, CD-HIT [ 25] was used to select up to maximum 300 most significant representative sequences.
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