A5-miseq is an updated pipeline designed to assemble microbial genomes from Illumina MiSeq data, addressing the complexity and accessibility issues of open-source bacterial genome assembly. The pipeline automates key steps such as adapter trimming, quality filtering, error correction, contig and scaffold generation, and misassemblies detection. It leverages long reads from MiSeq (up to 400nt) and uses read pairing information during contig generation, resulting in significantly improved assemblies compared to the original A5 pipeline. A5-miseq can produce high-quality assemblies with as little as 20-fold sequence data coverage on a laptop, achieving higher contiguity and completeness of reference genes. The pipeline is available under the GPL open-source license and has been benchmarked on the GAGE-B dataset, showing substantial improvements in assembly accuracy and efficiency. The authors recommend A5-miseq for researchers with limited bioinformatics experience or computing resources.A5-miseq is an updated pipeline designed to assemble microbial genomes from Illumina MiSeq data, addressing the complexity and accessibility issues of open-source bacterial genome assembly. The pipeline automates key steps such as adapter trimming, quality filtering, error correction, contig and scaffold generation, and misassemblies detection. It leverages long reads from MiSeq (up to 400nt) and uses read pairing information during contig generation, resulting in significantly improved assemblies compared to the original A5 pipeline. A5-miseq can produce high-quality assemblies with as little as 20-fold sequence data coverage on a laptop, achieving higher contiguity and completeness of reference genes. The pipeline is available under the GPL open-source license and has been benchmarked on the GAGE-B dataset, showing substantial improvements in assembly accuracy and efficiency. The authors recommend A5-miseq for researchers with limited bioinformatics experience or computing resources.