These approaches are limited to large organisms, as they require either direct visual inspection or electronic devices that can be attached by physical means to the body of the organism in question. For metazoa, a number of approaches have been utilized to track individuals of a species, including simple bands or markings conferring unique identifiers, Passive Integrated Transponders (PITs), telemetry devices, and biologgers. The barCoder algorithm facilitates the generation of synthetically barcoded biological simulants by (a) eliminating the task of creating modules by hand, (b) minimizing optimization of PCR assays, and (c) reducing effort wasted on non-unique barcode modules.ĭeveloping an understanding of organisms in their natural ecological niches requires the ability to measure the dynamic interaction with their environment, either at the level of the individual or at population scales. Real-time PCR detection assays directed at barcodes were specific and yielded minimal cross-reactivity with a panel of near-neighbor and potential contaminant materials. We tested the ability of our algorithm to suggest appropriate barcodes by generating 12 modules for Bacillus thuringiensis serovar kurstaki-a simulant for the potential biowarfare agent Bacillus anthracis-and three each for other potential target organisms with variable G+C content. Optimal amplification includes metrics such as ideal melting temperature and G+C content, appropriate spacing, and minimal stem-loop formation unique identification includes low BLAST hits against the target organism, previously generated barcode modules, and databases (such as NCBI). Each module is designed to yield optimal PCR amplification and unique identification. barCoder utilizes the genomic sequence of the target strain and a set of user-specified PCR parameters to generate a list of suggested barcode “modules” that consist of binding sites for primers and probes, and appropriate spacer sequences. Here we introduce barCoder, a bioinformatics tool that facilitates the process of creating sets of uniquely identifiable barcoded strains. Manual design of these barcodes is feasible for small sets, but expansion of the technique to larger pools of distinct and well-functioning assays would be significantly aided by software-guided design. Recent work has shown that genomic insertion of short identification tags, called “barcodes” here, allows detection of chromosomally tagged strains by real-time PCR. Tracking dispersal of microbial populations in the environment requires specific detection methods that discriminate between the target strain and all potential natural and artificial interferents, including previously utilized tester strains.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |