Computational protocol: The role of retinoic acid signaling in starfish metamorphosis

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Protocol publication

[…] To gather sequence of RA signaling related genes, we performed transcriptome analysis on samples of blastula (13 h post-fertilization), brachiolaria larva (14 dpf) and adult epidermis in starfish P. pectinifera. Each sample was collected from three biological replicates: The samples of blastula and brachiolaria larva were collected from progeny of three distinct pairs of parents, and the adult epidermis was collected from three mother specimens. Total RNA was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and treated with DNase and cleaned up using the RNeasy kit (Qiagen, Hilden, Germany). Paired-end libraries (200 bp) were prepared using a TruSeq RNA Sample Preparation Kit (Illumina). Sequencing on the Hiseq 2000 platform (Illumina, San Diego, CA, USA) was performed at the National Institute of Basic Biology (NIBB) The raw reads were deposited in the DDBJ Sequence Reads Archives (DRA006662). The low-quality reads were filtered using the NGS QC Toolkit57 []. De novo assembly was conducted by Trinity58 (version: trinityrnaseq_r20140717) [].We recovered nucleotide sequences coding for raldh, rar and rxr from gene models assembled from our de novo transcriptome data of larval stages for P. pectinifera. The orthologies of these genes were confirmed by molecular phylogenetic analysis (Additional file : Fig. S1, Additional file : Fig. S2). All sequences are shown in Additional file : Table S1 and aligned using MAFFT ver. 7 (https://mafft.cbrc.jp/alignment/server/) with the default parameters []. Amino acid sites for tree construction were selected by trimAl using a gap threshold value of 0.8 []. A best-fitting amino acid substitution model and a maximum likelihood tree were inferred using RAxML 8.2.0 []. Confidence values were calculated after 1000 bootstrap runs. […]

Pipeline specifications

Software tools NGS QC Toolkit, Trinity, MAFFT, trimAl, RAxML
Databases DDBJ
Application Phylogenetics
Organisms Caenorhabditis elegans
Diseases Amino Acid Metabolism, Inborn Errors