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A variation of BLAST called BLAST 2 Sequences can be used to find local alignments between any two protein or nucleotide sequences of interest (Tatusova and Madden 1999). Although the BLAST engine is used to find the best local alignment between the two sequences, no database search is performed. Rather, the two sequences to be compared are specified in advance by the user. The method is particularly useful for comparing sequences that have been determined to be homologous through experimental methods or for making comparisons between sequences from different species. Returning to the Protein BLAST (BLASTP) search page shown in Figure 3.6, checking the box marked “Align two or more sequences” will change the structure of the page, now allowing for the user to enter both the query and subject sequences that will be compared with one another (Figure 3.11). As with any BLAST search, the user can adjust the standard array of BLAST-related options, including the selection of scoring matrix and gap penalties. A sample of the results produced by the BLAST 2 Sequences method is shown in Figure 3.12, comparing the transcription factor SOX-1 from H. sapiens and the ctenophore Mnemiopsis leidyi, the earliest branching animal species dating back at least 500 million years in evolutionary time (Ryan et al. 2013; Schnitzler et al. 2014). The major difference between this output and the typical BLAST output is the inclusion of a dot matrix view of the alignment, or “dotplot.” Dotplots are intended to provide a graphical representation of the degree of similarity between the two sequences being compared, allowing for the quick identification of regions of local alignment, direct or inverted repeats, insertions, deletions, and low-complexity regions. The dotplot in Figure 3.12 indicates two regions of alignment, and additional information on those two regions of alignment is provided in the Alignments section at the bottom of the figure. As with all BLAST searches, the Alignments section provides the user with the usual set of scores, the E value, and percentages for identities, positives, and any gaps that may have been introduced.

Figure 3.11 Performing a BLAST 2 Sequences alignment. Clicking the check box at the bottom of the Enter Query Sequence section expands the search page, generating a new Enter Subject Sequence section. Here, sequences for the transcription factor SOX-1 from human and the ctenophore Mnemiopsis leidyi have been used as the query and subject, respectively (Schnitzler et al. 2014). Here, only the BLASTP algorithm is available in the Program Selection section, as a one-to-one alignment has already been specified. The usual set of algorithm parameters is available, allowing the user to fine-tune the alignment as needed.