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Hidden paralogy is rare in single-copy gene families selected as likely orthologs (orthology establishment) under application of the reciprocal hit criterion [31].
The putative orthologous pairs were defined based on the reciprocal hit criterion and then analyzed with the program MCL, which utilizes Markov Clustering (MCL) by creating a similarity matrix from e-values and then clustering proteins into related families.
To better understand the evolution of this family, we extracted a large set of TGFβ homologues from available animal genome sequences and identified those that had a human inhibin/activin gene as a top hit (this corresponds to the bidirectional BLAST best hit criterion) (Fig. S2A, Table S1).
Parameters were set as "default" except that the best hit criterion used was "bidirectional".
To ascertain that we identified class 4 HDACs, we first used the 'reciprocal best BLAST hit' criterion.
Nevertheless, they are the closest relatives to the mouse Jmjd6 as defined by the best reciprocal BLAST hit criterion.
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Based on best BLASTX hit criteria that was used previously for barley, these probe sets matched 583 unique rice loci.
Electrophysiological measurements confirmed a hit rate at or above 17%% when the relatively stringent hit criteria of greater than 80%% channel inhibition at 10 μ M was applied.
Genes that satisfied BLASTP alignment threshold (35% sequence identity over 80% length of the smallest protein) or bidirectional best hit criteria, were defined as putative orthologs.
They were identified through searches on non-redundant NCBI protein database (September 2008, see Additional file 2 (information A2 for details of search parameters and hit criteria).
These chains can represent both orthologous and paralogous sequences, but are typically filtered with alignment score thresholds or 'best hit' criteria to retain primarily orthologous sequences.
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