In silico analysis of Mi-col-5

The amino acid sequence of Mi-col-5 was deduced from the nucleotide sequence using expasy translate tool (http://web.expasy.org/translate/). The amino acid and nucleotide sequences of Mi-col-5 were analyzed using NCBI blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Physical and chemical parameters of the predicted amino acid sequence were computed using ProtParam (Gasteiger et al. 2005). Clustal Omega was used for alignment between amino acid sequences of Mi-col-5 and Mj-col-5 (Sievers et al. 2011). Domain architecture analysis was done using SMART and MOTIF search (Letunic et al. 2015). SOPMA was used for the analysis of secondary structure of the predicted amino acid sequence of Mi-col-5 (Combet et al. 2000). Multiple sequence alignment of the deduced amino acid sequence with cuticle collagen proteins identified in M. incognita and other species was done using Clustal Omega (Roy et al. 2010). A phylogenetic tree was constructed by maximum likelihood method using MEGA6 (Tamura et al. 2013). Intrinsic folding and unfoldability of Mi-col-5 was predicted using Fold Index (Prilusky et al. 2005). Prediction of transmembrane domain for Mi-col-5 and other cuticle collagen proteins was performed using TMHMM server V. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), and signal peptide prediction was performed through SignalP 4.1 server (Petersen et al. 2011). Subcellular localisation predictor CELLO v2.5 server (Yu et al. 2006) was used for prediction of subcellular localization of Mi-col-5. Three-dimensional structure of the protein Mi-col-5 was predicted by using I-TASSER server (Roy et al. 2010; Yang et al. 2015). The predicted model was evaluated using PROCHECK server (Laskowski et al. 1996).