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In order to identify the complete set of T6SS components in all the species of each organism, each of the T6SS component identified in a particular species of an organism was then used to search for their orthologs using a second blast search against its other species for which complete genome sequences were known.
Our bioinformatics strategy, using a second BLAST stage to detect these cases worked well overall, but a number of such cases did escape the initial automated analysis, and have been corrected by manual merging of alignments.
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The CTL cell lines were generated using a second stimulation with irradiated, autologous PHA blasts pulsed with the reactive cA1 peptide.
These 240 best-hit yeast proteins were then used as the query in a second BLAST against the entire human proteome.
A second BLAST search used NCBI's yeast database (yeastDB) [ 39], which is a single curated set of Saccharomyces cerevisiae protein sequences available at the NCBI's RefSeq database.
Instead of then taking the LCA of the retained matches, a second BLAST search is done using the best BLAST match as the query, and all sequences above the p threshold plus the original query sequence as the new reference.
For detecting distant homologs we used sequences from 25 organisms as seeds for a second BLAST search.
First, using a self-BLAST and an SGN Perl script (precluster.pl), we implemented a pre-clustering phase of the EST sequences with a minimum identity percentage of 90% and a minimum alignment length of 30 bp.
A fourth blast near a cinema killed one person.
For the first we use a blast furnace, the second a rolling mill.
It appears the bombers used the first blast to lure police to the scene before setting off other explosives.
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