Computational protocol: RuBisCO in Non-Photosynthetic Alga Euglena longa: Divergent Features, Transcriptomic Analysis and Regulation of Complex Formation

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Protocol publication

[…] Homologs of the investigated proteins (RBCL, RBCS, RuBisCO activase RCA, and RuBisCO assembly chaperone RAF) were identified by BLAST [] in the non-redundant protein database at NCBI, in transcriptome assemblies of relevant species generated by the Marine Microbial Eukaryote Transcriptome Sequencing Project ( [], and in our unpublished transcriptome of E. longa obtained by assembling RNA-seq Illumina reads from two differently treated cultures (details on the RNA preparation, RNA-seq, and transcriptome assembly will be published elsewhere). The cDNA sequences corresponding to RCA and RBCS proteins from E. longa were deposited at GenBank with accession numbers KT818573-KT818576. Accession numbers of all sequences of RBCL, RBCS, and RCA proteins analyzed in this study are listed in , , and Tables, respectively. Sequences were aligned using MAFFT 7 (Multiple Alignment using Fast Fourier Transform) []. The alignment was manually refined using BioEdit 7.1.7 and ambiguously aligned positions were removed []. The resulting RBCL alignment contained 53 sequences and 473 amino acid positions, the RBCS alignment contained 42 sequences and 122 amino acid positions, and the RCA alignment contained 30 sequences and 301 amino acid positions. Maximum likelihood (ML) trees were inferred from the alignments using RAxML 8.1.11 employing the strategy of rapid bootstrapping followed by a "thorough" ML search on the original dataset with the LG+Γ substitution model (1,000 bootstrap replicates) []. In addition, a Bayesian phylogeny was inferred using PhyloBayes 3.3b with the following parameters: 15,000 generations under the C20 model with Poisson exchange rate, sampling every 100 generations, and maximum divergence allowed set to 0.1 []. […]

Pipeline specifications

Software tools MAFFT, BioEdit, RAxML, PhyloBayes
Application Phylogenetics
Organisms Euglena longa, Euglena gracilis
Chemicals Cycloheximide