Complete mitogenome draft assembly using a BLAST–CAP3 web workflow: Gryllus bimaculatus as a model

Abstract

Introduction: Mitochondrial genomes recovered from public sequencing repositories offer cost-effective molecular resources without additional laboratory work. Building on a previously validated targeted Basic Local Alignment Search Tool–Contig Assembly Program version 3 (BLAST–CAP3) workflow for single-gene assembly, the present study demonstrates its extension to complete mitochondrial genome draft assembly using a publicly available whole-genome sequencing (WGS) dataset of Gryllus bimaculatus. Materials and methods: Mitochondrial reads were retrieved using custom National Center for Biotechnology Information (NCBI) nucleotide Basic Local Alignment Search Tool (BLASTN) parameters against the published G. bimaculatus mitochondrial genome reference, followed by CAP3 de novo assembly on Galaxy Europe. Results: Assembly yielded a single complete contig of 15,954 base pairs (bp) with 99.574% nucleotide identity to the reference (E-value = 0.0; bit score = 29,085). Independent read mapping validation confirmed that all 15,954 positions of the assembled contig were covered by at least 2× read depth (mean 44.5×; maximum 92×), with 4731 of the 4749 deduplicated reads (99.6%) successfully mapped. A critical obstacle, failure of standard CAP3 parameters in the A+T-rich control region (~1240 bp) due to the tandem repeat architecture, was resolved by reducing the overlap length cutoff to 10–15 bp. Self-dotplot analysis confirmed that tandem repeats were confined to positions ~14,650–15,450 bp with a repeat unit spacing of ~150–200 bp. Conclusions: This study documents a practical and potentially transferable solution for animal mitochondrial genome draft assembly from public sequencing data. The complete pipeline requires no local software installation and is executable using web-based tools within a single working day.