Computational protocol: Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes

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Protocol publication

[…] The HMM search program in the HMMER package (version 3.1b1) [] was used to detect enolase candidates. HMM profiles of the enolase N-terminal domain (Enolase_N, PF03952) and C-terminal domain (Enolase_C, PF00113) in the Pfam 27.0 database [] were queried against deduced protein sequences in a B. mori Ensembl Gene dataset [] and a cDNA dataset [] with default parameters.A search for enolase orthologs among the genes of the following species was conducted using BLAST methods: D. melanogaster, M. sexta, A. gambiae, A. mellifera, T. castaneum, and H. sapiens. Global homology searches were conducted using Genetyx ver. 10 (Genetyx Co. Ltd., Tokyo, Japan). A phylogenic analysis was conducted using MEGA ver. 7 []. A protein motif search was conducted using SMART (http://smart.embl-heidelberg.de/). The alignment of the BmEno amino acid sequences and enolase orthologs from other species was conducted using CLC Sequence viewer 7.6.1 (CLC Bio Japan Inc. Tokyo, Japan). All analyses were performed with default parameters for the software. [...] To quantify RNA expression levels, 1 μg of total RNA from pooled testis tissue dissected from day 0 fifth-instar larvae to day 0 adults (n = 3 each) was used for cDNA synthesis. qRT-PCR was performed in a 20 μl reaction volumes with 0.5 μl of the cDNA template and primers (Table ) with a KAPA SYBR Fast qRT-PCR Kit (Nippon Genetics Co., Ltd., Tokyo, Japan) in accordance with the manufacturer’s instructions. qRT-PCR was performed on a Step One plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) following the Delta-Delta Ct method. Ribosome protein 49 (GeneID: 778453) was used as an endogenous reference for the standardization of RNA expression levels, and all data were calibrated against universal reference data. Relative quantification (RQ) values represent the relative expression level against a reference sample. All samples were assayed in triplicate as technical replications. [...] Total RNA was isolated from the testis of day 3 fifth-instar larvae of the B. mori o751 wild type strain using a PureLink® RNA extraction kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s protocol. The quality of RNA was assessed using an Agilent Bio-analyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing cDNA libraries were constructed with 4 μg of total RNA from o751 wild type testis samples (n = 3) with a Truseq RNA Sample Preparation Kit Set A (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol. RNA-seq was performed using a HiSeq 2500 system (Illumina Inc.). The data quality of the fastq files was verified with the FastQC tool (Babraham Bioinformatics, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The 44 M paired-end reads (2 × 150 bp) were mapped to the reference B. mori genomes available in the Ensembl Genome database [, ] using the Tophat program version 2.0.13 with default parameters []. BAM formatted files generated by Tophat were sorted and indexed using SAMtools [] and then converted to Wiggle track format (WIG) files using the bam2wig software (https://github.com/MikeAxtell/bam2wig). This allowed us to visualize the density of reads mapped to the specific region of interest. Histograms of mapped reads were generated using the Spotfire Cloud software with TIBCO Spotfire’s “Better World” program license (TIBCO Software, Inc., Palo Alto, CA, USA) (http://spotfire.tibco.com/better-world-donation-program/). […]

Pipeline specifications

Software tools HMMER, MEGA, CLC Sequence Viewer, GeneID, FastQC, TopHat, SAMtools, bam2wig
Databases Pfam
Applications Phylogenetics, RNA-seq analysis
Organisms Bombyx mori
Diseases Ovarian Neoplasms
Chemicals Amino Acids