biostudies-literatureWagner ADUniversity of Mississippi Provost’s officeUniversity of Mississippi Provost's officeDepartment of BioMolecular SciencesNational Institutes of HealthNIGMS NIH HHSNIH HHSNational Institute of General Medical Sciences as Centers of Biomedical Research Excellencejkaf135https://www.ebi.ac.uk/biostudies/studies/S-EPMC12341861biostudies-literatureUnknown15(8)Extracting DNA from cyanobacteria can be a challenge because of their diverse morphologies, challenging cellular structure, and the heterotrophic microbiome often present within cyanobacterial cultures. As such, even with high DNA yields, the percentage of reads coming from the cyanobacterial host can be low, leading to an incomplete cyanobacterial genome assembly. In this research, we optimized a DNA isolation protocol using three iterative cell lysis steps to enrich the portion of DNA isolated coming from the cyanobacterial host rather than the heterotrophic microbiome. In order to utilize in-house nanopore sequencing, we faced a challenge using our lysis protocol: the iterative lysis approach led to more DNA shearing than is ideal for this sequencing technology. To solve this, we used tG3 (Bethesda, Md.)Using repeated lysis steps fractionates between heterotrophic and cyanobacterial DNA extracted from xenic cyanobacterial cultures.PMC12341861P30 GM122733P30GM122733P20GM130460P20 GM130460Starks VABoudreau PDAhmed MMAWagner ADfalseUsing repeated lysis steps fractionates between heterotrophic and cyanobacterial DNA extracted from xenic cyanobacterial cultures.Extracting DNA from cyanobacteria can be a challenge because of their diverse morphologies, challenging cellular structure, and the heterotrophic microbiome often present within cyanobacterial cultures. As such, even with high DNA yields, the percentage of reads coming from the cyanobacterial host can be low, leading to an incomplete cyanobacterial genome assembly. In this research, we optimized a DNA isolation protocol using three iterative cell lysis steps to enrich the portion of DNA isolated coming from the cyanobacterial host rather than the heterotrophic microbiome. In order to utilize in-house nanopore sequencing, we faced a challenge using our lysis protocol: the iterative lysis approach led to more DNA shearing than is ideal for this sequencing technology. To solve this, we used t2025-01-01T00:00:00Z2025 Aug2026-05-29T16:52:32.728Z2026-04-08T05:27:29.452ZS-EPMC123418614049859210.1093/g3journal/jkaf135