Computational protocol: Identification of Candidate Odorant Receptors in Asian Corn Borer Ostrinia furnacalis

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Protocol publication

[…] Male and female antennae were dissected from 2-day-old adults, and frozen in liquid nitrogen. RNA was immediately isolated from the frozen antennae using the QuickGene RNA tissue Kit SII (RT-s2; KURABO, Neyagawa, Japan). The antennae from more than 20 individuals were pooled for a single RNA isolation experiment. Three biological repeats for each sex were made for the analysis of expression levels. Sequencing libraries were prepared using the TruSeq RNA Sample Preparation Kit v2 according to the LS protocol of the manufacturer’s instructions (Illumina, Inc., San Diego, CA, USA) using 1 μg of total RNA from each sample, except for the following modifications to select the library with long inserts. Incubation time of purified mRNA fragmentation was changed from 8 min to 30 sec at 94°C, and 0.7×volume of the AMPure XP beads was used in the all purification steps. Prepared libraries were mixed at a concentration identical to each other in a 1.5 ml tube and applied for cluster generation on the MiSeq system using the MiSeq Reagent Kit v3 (Illumina, Inc., San Diego, CA, USA). A total of 6 libraries were indexed and applied for a single multiplex run in the 300 bp single-end mode. The raw data were deposited in the DDBJ Sequence Read Archive under accession number DRA002255. The reads were preprocessed with cutadapt v1.2.1 [] for quality trimming at QV30 with a minimum length of 50 bp. The pass-through reads were pooled and assembled using Trinity r2013_08_14 (http://trinityrnaseq.sourceforge.net/) []. Open reading frames were extracted from the Trinity contigs with TransDecoder (http://transdecoder.sourceforge.net/) [, , , ] using the script that came with the Trinity distribution without modification. [...] Phylogenetic relationships of O. furnacalis odorant receptors (OfurORs) were analyzed against BmorORs and Cydia pomonella odorant receptors (CpomORs) [, ]. A total of 164 amino acid sequences were aligned using MAFFT v7.130 with the option E-INS-i []. Phylogenetic relationship was deduced by the maximum likelihood method using RAxML v8.0.17 [, ] with the GAMMA model for rate heterogeneity and the WAG model for substitution matrix. In addition, the rapid hill-climbing search algorithm (–f d) was used. Model optimization precision in log likelihood units for final optimization of tree topology (–e) was set at 0.0001. The tree image was created using FigTree v1.4.1 (http://tree.bio.ed.ac.uk/software/figtree) []. […]

Pipeline specifications

Software tools MAFFT, RAxML, FigTree
Application Phylogenetics
Organisms Ostrinia furnacalis, Homo sapiens