A Targeted BLAST‐CAP3 Workflow for Rapid and Reproducible Mitochondrial Gene Assembly From Public Transcriptomes: Acheta domesticus as a Model

Rapid access to mitochondrial marker genes is essential for species identification and phylogenetics, but conventional barcoding or genome skimming remains time‐consuming and resource‐intensive. Public RNA‐seq archives, an underused source of mitochondrial transcripts, can be leveraged through an ultralight, fully web‐accessible pipeline that retrieves and assembles the complete cytochrome c oxidase subunit I (COX1) gene. Seven public Acheta domesticus RNA‐seq datasets (SRR7692599–SRR7692605) were retrieved from the NCBI Sequence Read Archive. A targeted BLAST‐based filtering approach was applied to extract COX1‐related reads, which were then assembled using the CAP3 tool on Galaxy Europe. Assembly quality was assessed by ClustalW alignment and confirmed by BLAST validation against the NCBI nucleotide database. Between 180 and 214 COX1‐related reads (mean 200.9 ± 11.8) were recovered per dataset. CAP3 produced complete or extended COX1 contigs (1545–1784 bp; up to 116.5% of the 1531 bp coding length due to flanking UTR‐like sequence). All seven assemblies showed 100% nucleotide coverage to the reference after orientation correction. No evidence of off‐target mitochondrial genes or nuclear mitochondrial pseudogene (NUMT)‐like matches was observed among the top BLASTN hits for the assembled contigs. The complete workflow was completed in approximately 1 h per dataset (from BLAST‐based read extraction to final BLAST validation) while producing near full‐length COX1 contigs. Robustness was further supported by successful COX1 recovery from additional cricket species transcriptomes and a whole‐genome sequencing dataset using the same parameters. This streamlined pipeline enables recovery of mitochondrial genes from public transcriptomic resources and can be adapted for other highly expressed genes. It provides a cost‐effective alternative to traditional molecular methods. Key Points A rapid and reproducible transcriptome‐based workflow is developed for mitochondrial COX1 gene assembly from RNA‐Seq data. BLAST‐based targeted read extraction combined with CAP3 de novo assembly enables efficient recovery of full‐length gene sequences. Assembly validation using ClustalW and BLASTn confirms 100% nucleotide coverage and high sequence accuracy. The pipeline is species‐independent, web‐accessible, and adaptable to other mitochondrial protein‐coding genes and diverse sequencing datasets.