Sequencing traces were analysed using Sequencher software (GeneCodes Corporation, Ann Arbor, MI, USA), and multiple sequences aligned to aid mutation detection.
Predicted annotations (tab-separated text files), trees (newick files), and multiple sequence alignments (aligned FASTA files) can be downloaded for further offline analysis.
An alternative method is structure-based multiple sequence alignment which aligns three-dimensional protein structures and then aligns sequences progressively to the initial structure alignment [22].
These results are derived from a 513 188 amino acid concatenated, gap-free, multiple sequence alignment (~250 aligned amino acid positions per gene) produced from the 2 135 triplets possessing at least a 50 amino acid aligned block.
All sequences were aligned by using the multiple sequence alignment editor ALIGN-IR (LI-COR), and consensus sequences for every gene sequenced were determined.
The QSOX multiple sequence alignment was then aligned to the atomic coordinate file using the ConSurf server [ 30], resulting in a color coded scheme of conserved residues mapped onto the model of the H. sapiens QSOX1 closed state structure.
We used a multiple sequence alignment to align and subsequently encode the sequences with five different descriptors, namely the hydrophobicity scale of Kyte and Doolittle [ 12], molecular weight, isoelectric point, pKa and HIV-1 cleavage probability [ 13].
For comparison of the primary structures of biomineralization proteins and their families and groups, the multiple sequence alignment mode "Align" was used, implemented through "ClustalW 2.0.12" (with the mode settings not configured).
Sequences with significant E-value similarity to CCDC147 where kept, and the G. magnirostris and T. guttata (and later G. fortis) CCDC147 predicted sequences were added to multiple sequence alignments that were aligned using T-Coffee [ 89].
COACH (Comparison of Alignments by Constructing Hidden Markov Models) aligns two multiple sequence alignments by constructing a profile HMM from one alignment and aligning the other to this HMM; it is freely available as a stand-alone version.
