IMAGE PROCESSING OF DNA NANOBARCODE

Master of Engineering ThesisAn image processing software package was designed to assign clearly distinguishable colors to DNA nanobarcode probes labeled with varying ratios of red and green fluorescent dyes. The DNA nanobarcode probes were produced using novel tri-strand Y-shaped DNA secondary structures. These Y-shaped DNA structures are multivalent and anisotropic, allowing for specific and controlled hybridization to other Y-shaped DNA building blocks to create a dendrimer-like DNA structure (DL-DNA). Here, the image processing of fluorescence-intensity-encoded nanobarcodes was explored using the MATLAB software environment. DNA nanobarcodes were created with varying green:red intensity ratios: 4G1R (4 green : 1 red), 2G1R, 1G1R, 1G2R and 1G4R. Additionally, these nanobarcodes incorporated specific molecular probes to target the DNA of bacillus anthracis (anthrax), francisella tularensis (?rabbit fever?), Ebola virus, a positive control and corona virus (SARS) respectively. Fluorescence was amplified by attaching DL-DNA to polystyrene beads through biotin-avidin interactions. The nanobarcodes were visualized using a fluorescent microscope and pseudocolor images were obtained. These images were easy to distinguish for 4G1R (bright green) and 1G4R (bright red), but were difficult to distinguish for other ratios which showed up as different shades of yellow, green and orange. Using the image processing software, these nanobarcodes were assigned highly distinguishable colors based on their green:red intensity ratio, allowing for easy and fast visual identification of the pathogens targeted. With further development, this software package will be able to assign at least 25 different and visually distinguishable colors for two and three fluorescent dyes arranged in varying ratios to create high throughput visual screening of harmful pathogens.Cornell University, Molecular Bioengineering Laborator

The genital microbiome and its potential for detecting sexual assault

Since its inception, the Human Microbiome Project (HMP) has provided key discoveries that can be applied to forensics, in addition to those of obvious medical value. Whether for postmortem interval estimation, geolocation, or human identification, there are many applications of the microbiome as an investigative lead for forensic casework. The human skin microbiome has shown great potential for use in studies of transfer and human identification, however there has been little focus on the genital microbiome, in particular penile skin which differs from other body sites. Our preliminary data on both the penile and vaginal microbiome demonstrates potential value in cases of sexual assault. In this study we describe genital microbial signatures based on the analysis of five male and five female genital samples and compare these results to those from longitudinal studies. Selected taxa, e.g., Gardnerella, Lactobacilli, Finegoldia, Peptoniphilus , and Anaerococci, are shown to be candidate constituents of the genital microbiome that merit investigation for use in sexual assault casework

Microbial diversity, genomics, and phage–host interactions of cyanobacterial harmful algal blooms

ABSTRACT The occurrence of cyanobacterial harmful algal blooms (cyanoHABs) is related to their physical and chemical environment. However, less is known about their associated microbial interactions and processes. In this study, cyanoHABs were analyzed as a microbial ecosystem, using 1 year of 16S rRNA sequencing and 70 metagenomes collected during the bloom season from Lake Okeechobee (Florida, USA). Biogeographical patterns observed in microbial community composition and function reflected ecological zones distinct in their physical and chemical parameters that resulted in bloom “hotspots” near major lake inflows. Changes in relative abundances of taxa within multiple phyla followed increasing bloom severity. Functional pathways that correlated with increasing bloom severity encoded organic nitrogen and phosphorus utilization, storage of nutrients, exchange of genetic material, phage defense, and protection against oxidative stress, suggesting that microbial interactions may promote cyanoHAB resilience. Cyanobacterial communities were highly diverse, with picocyanobacteria ubiquitous and oftentimes most abundant, especially in the absence of blooms. The identification of novel bloom-forming cyanobacteria and genomic comparisons indicated a functionally diverse cyanobacterial community with differences in its capability to store nitrogen using cyanophycin and to defend against phage using CRISPR and restriction-modification systems. Considering blooms in the context of a microbial ecosystem and their interactions in nature, physiologies and interactions supporting the proliferation and stability of cyanoHABs are proposed, including a role for phage infection of picocyanobacteria. This study displayed the power of “-omics” to reveal important biological processes that could support the effective management and prediction of cyanoHABs.IMPORTANCECyanobacterial harmful algal blooms pose a significant threat to aquatic ecosystems and human health. Although physical and chemical conditions in aquatic systems that facilitate bloom development are well studied, there are fundamental gaps in the biological understanding of the microbial ecosystem that makes a cyanobacterial bloom. High-throughput sequencing was used to determine the drivers of cyanobacteria blooms in nature. Multiple functions and interactions important to consider in cyanobacterial bloom ecology were identified. The microbial biodiversity of blooms revealed microbial functions, genomic characteristics, and interactions between cyanobacterial populations that could be involved in bloom stability and more coherently define cyanobacteria blooms. Our results highlight the importance of considering cyanobacterial blooms as a microbial ecosystem to predict, prevent, and mitigate them