BigDye-terminator sequencing has a very low error rate. Nevertheless, our rule-of-thumb is to require 10 BigDye-terminator reads (~ 3% of the sequence reads) to securely detect a bacterium. Our molecular probe technology requires a reasonably secure PF-6463922 chemical structure genome sequence for each bacterium and the synthesis of long oligonucleotides. Second generation sequencing is providing
bacterial genome sequences faster and cheaper than BigDye-terminator sequencing. The cost of synthesizing oligonucleotides is coming down, while the length is going up. For the molecular probes, the Homers are based upon single copy sequences. Thus, unlike rDNA-based detection, there is no copy number variation among bacterial GS-9973 research buy genomes that could confound the results. However, to design the Homers, we started with complete genome sequences of specific strains of any given bacterial species. The bacterial genome sequence section of GenBank
(presumably) contains only a fraction of the genome sequences of all of the strains for any given species. Thus, a molecular probe may be correctly positive for one strain’s genome and correctly negative for another’s. This situation would give rise to false negatives in detecting bacteria. We have attempted to minimize this possibility by employing multiple probes per genome and with Homers derived from different parts of the genome sequence. We have employed GF120918 in vitro two very different assays for the molecular probes: Tag4 array and SOLiD sequencing. There was an apparent lack of good, relative quantitation for both assays, as seen for the simulated clinical samples. With the Tag4 assay, fluorescence intensity is an exponential function of mass and, thereby, inherently difficult to quantitate.
However, the assay for each sample requires an individual Tag4 array, and, therefore, each Tag4 assay is independent of the other Tag4 assays. The SOLiD assay requires only counting many the number of reads supporting the presence of each bacterium. However, as with any multiplex sequencing, the samples are not independent, as there is a limit to the total number of reads. Our goal is to produce a technology that will detect bacteria without culture, with commercially available reagents, highly multiplexed, and that will ultimately be fast and inexpensive. Other investigators have invented or adapted technologies toward likely the same goal. Several examples follow. The Insignia system is closest to our technology [13, 14]. The system is in two parts. The first part is the publically available software that defines oligonucleotides unique to the target genome of interest . The second part is a quantitative PCR assay (qPCR) . The software is definitely useful. The qPCR assay cannot be multiplexed. Nikolaitchouk et al.  applied “”checkerboard DNA-DNA hybridization”" to detect the microbes in the human female genital tract and achieved a 13-plex reaction.