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Background Global estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately contribute new information to shape public policy response on cholera control and elimination. Methodology/Principal findings Whole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor. Conclusions WGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers. This provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas., Author summary Cholera affects more than 4 million people globally every year; people predominantly living in poverty or in resource-constrained conditions including political crises or natural disasters. Cholera’s typical presentation is characterized by rapid onset of acute watery diarrhea and vomiting which can progress from watery stool to shock in as little as four hours. Laboratory conditions needed for culture confirmation and strain preservation are rarely to never present in these affected areas. In fact, many cholera endemic areas in Sub-Saharan African are so remote that even treatment response alone is often challenging. Here we present the genomic analysis of DNA extracted from dried filter paper, which is a low-cost, low-tech and sustainable method. Previously this method has facilitated cholera confirmation by PCR, but we demonstrate that this method is also suitable for whole genome sequencing and subsequent strain characterization by presenting the analysis of samples from an outbreak in a remote area of Cameroon. This method will facilitate the understanding of the molecular epidemiology in cholera-prone areas, which were previously too challenging to attempt. It also introduces a method that can be used on a broader scale for diarrheal disease surveillance, including providing a window into co-infection and microbiome analyses.