Project description:To evaluate targeted MinION next generation sequencing as a diagnostic method for detection of pathogens in human blood and plasma, human blood or plasma samples were spiked with measured amounts of viruses, bacteria, protozoan parasites or tested pathogen-free as negative controls. Nucleic acid was extracted from samples and PCR amplification performed in multiplex primer pools with a procedure described in ArrayExpress experiment submission ID 18379. The PCR products were used for library preparation. The libraries sequenced on an Oxford Nanopore MinION. The passed reads aligned with a custom reference file to determine the identity of the pathogen in the sample.
Project description:A survey and analytical assessment of the results of fundamental works on studying blood metagenome (set of all non-human DNA) is carried out. All works on determining bacterial DNA concentration in the whole blood of healthy people are reviewed. Detailed comparison of characteristics of 16S rRNA test (hereinafter 16S-test) and whole metagenome sequencing test (hereinafter WMS-test) is carried out and published in Supplement S1. One of main goals of this review is to identify the drawbacks and mistakes which the studied works contain, particularly to emphasize the crucial importance of determining total concentration of bacterial DNA for comparing patients' metagenomes with those of healthy people as well as for comparing patients' metagenomes with each other. Controlling the level and composition of contamination is equally important. The absence of high-quality contamination control at each step (or at certain steps) of the research significantly reduces the reliability of achieved results. The given review is the first attempt to analyze and systematize the results of blood metagenome studies, whose number has increased considerably in the last few years. The review has been carried out as part of preparation for implementing a project on complex studying metagenomes of whole blood and skin biopsies of psoriatic patients.