The resulting matched and validated homologues, paralogues and orthologues were used for the multiple alignment comparison.
In order to evaluate the similarity level between SP212 and related proteins, we performed a multiple alignment comparison with a selected group of SPs and SPHs.
Fifty-five putative Cx. quinquefasciatus ORs were omitted (see Table S1) as they were likely incomplete and/or looked to be partially wrongly annotated sequences when subjected to multiple alignments comparison.
Based on the homology of 16S rRNA sequence, strains with different sequence similarity from relative species were selected, and multiple sequence alignment comparison was carried out by BioEdit program.
Multiple alignments for comparison and phylogenetic studies were made with the ClustalW [ 53] respecting the reading frame.
Because Murasaki is using different spaced seeds than BLASTZ and requires that seeds match in all input sequences (unlike TBA where less similar matches are introduced during progressive multiple alignment), a direct comparison of individual anchors between Murasaki and TBA is not helpful as we would not expect them to find the same anchors.
Multiple alignment and phylogenetic comparison of the individual proteins encoded by each of the fimbrial clusters revealed a higher variation among the major fimbrial subunits compared to the other structural proteins [see Additional file 2: Figure S1, Additional file 3: Figure S2, Additional file 4: Figure S3].
We also tested another alternative from the TBA package called Roast which appears to implement the method described in [23] which builds a multiple alignment based on pairwise comparisons between a reference sequence and all other sequences, thus in theory requiring time linear with respect to the number of sequences, similar to Murasaki.
The validity of these predictions was further enhanced by comparison, based on multiple alignment and structural information, to published orthologous counterparts.
Sequence assembling was performed with ContigExpress (VectorNTI©-software) and multiple alignment was performed by log-expectation comparison, using the MUSCLE (v.3.6) software [ 13].
