Computational protocol: Critical Early Roles for col27a1a and col27a1b in Zebrafish Notochord Morphogenesis, Vertebral Mineralization and Post-embryonic Axial Growth

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Protocol publication

[…] Fibrillar collagen sequences from human, mouse, zebrafish, chicken, and sea squirt were aligned with CLUSTALX2.0, and the conserved region of the C-propeptide was manually selected. MrBayes version 3.0 was used to generate Bayesian inference topologies . Analyses were run for 106 generations with tree samples every 100 generations by which point the average standard deviation of split frequencies had dropped below 0.1. We plotted likelihood values against generation number to determine when likelihood values had stabilized. The first 4400 trees were discarded as ‘burn-in’ before the values stabilized, and a consensus tree was generated with Bayesian posterior probability values. A full heuristic parsimony analysis with 1000 bootstrap replicates was also performed using PAUP*4.0b10 (data not shown). The tree generated from this analysis was identical to the Bayesian tree with the exception that in the full heuristic parsimony analysis, the col27a1 clade was basal to all other collagens of the in-group. The following GenBank sequences were used: Homo sapiens COL1A1 NP_000079, COL1A2 NP_000080, COL2A1 NP_001835, COL3A1 NP_000081, COL5A1 NP_000084, COL5A2 NP_000384, COL5A3 NP_056534, COL11A1 NP_001845, COL11A2 NP_542411, COL24A1 NP_690850, COL27A1 NP_116277; Mus musculus Col1a1 NP_031768, Col1a2 NP_031769, Col2a1 NP_112440, Col3a1 NP_034060, Col5a1 NP_056549, Col5a2 NP_031763, Col5a3 NP_058615, Col11a1 NP_031755, Col11a2 NP_034056, Col24a1 NP_082046, Col27a1 NP_079961; Danio rerio col1a1 NP_954684, col1a2 NP_892013, col1a3 NP_958886, col2a1a NP_571367, col11a1 NP_001073461, col27a1a GQ229459, col27a1b EF032484; Gallus gallus col1a1 P02457, col1a2 P02467, col2a1 NP_989757, col3a1 P12105, col5a1 NP_990121, col5a2 XP_421846, col11a1 XP_001231624, col24a1 XP_422363; Ciona intestinalis CiFCol2 AK113159. Since sequence data was unavailable for Danio rerio col5a2 and Gallus gallus col27a1, protein sequence predictions, ENSDARP00000041806 and ENSGALP00000011258, respectively, were used. […]

Pipeline specifications

Software tools Clustal W, MrBayes, PAUP*
Application Phylogenetics
Organisms Danio rerio, Homo sapiens