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Vibrios are ubiquitous marine bacteria that have long served as models for heterotrophic processes and have received renewed attention because of the discovery of increasing numbers of facultatively pathogenic strains. Because the occurrence of specific vibrios has frequently been linked to the temperature, salinity, and nutrient status of water, we hypothesized that seasonal changes in coastal water bodies lead to distinct vibrio communities and sought to characterize their level of differentiation. A novel technique was used to quantify shifts in 16S rRNA gene abundance in samples from Barnegat Bay, N.J., collected over a 15-month period. Quantitative PCR (QPCR) with primers specific for the genus Vibrio was combined with separation and quantification of amplicons by constant denaturant capillary electrophoresis (CDCE). Vibrio populations identified by QPCR-CDCE varied between summer and winter samples, suggesting distinct warm-water and year-round populations. Identification of the CDCE populations by cloning and sequencing of 16S rRNA genes from two summer and two winter samples confirmed this distinction. It further showed that CDCE populations corresponded in most cases to ϳ98% rRNA similarity groups and suggested that the abundance of these follows temperature trends. Phylogenetic comparison yielded closely related cultured and often pathogenic representatives for most sequences, and the temperature ranges of these isolates confirmed the trends seen in the environmental samples. Overall, this suggests that temperature is a good predictor of the occurrence of closely related vibrios but that considerable microdiversity of unknown significance coexists within this trend.The genus Vibrio encompasses a diverse group of heterotrophic marine bacteria including many facultative symbiotic and pathogenic strains. The latter include Vibrio cholerae, the causative agent of cholera, and V. parahaemolyticus and V. vulnificus, which together are responsible for most cases of fatal seafood poisoning (31). Vibrio infections are not limited to humans, as recently highlighted by reports of Vibrio species capable of killing coral tissue (3, 27), and vibrios represent a major source of concern in aquaculture facilities and marine aquaria (11,40,47). Because all of these pathogens appear to maintain planktonic populations, considerable interest exists in understanding the prevalence and dynamics of specific Vibrio populations in the environment.Most studies on Vibrio ecology to date have focused on specific members of the genus, leading to an extensive body of literature on their genetics and ecology. However, the diversity and dynamics of co-occurring Vibrio populations have only rarely been addressed (e.g., see references 1a, 4, 8, 17, 18, and 38) and more rarely still by using quantitative culture-independent methods (8,17,18,38). All quantitative surveys (by molecular techniques) have confirmed the ubiquity of vibrios but have, with the exception of one study (38), also suggested that Vibrio populations are generally Ͻ1% ...

ObjectiveTo develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).MethodsTwo hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths.ResultsAt the lowest sequencing depth, corresponding to 204 000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410 000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620 000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches.ConclusionDigital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.