Project description:Background: The analysis of oligonucleotide microarray data in pathogen surveillance and discovery assays is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied. Results: An examination of both positive predictive value and false positive rates was employed to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney U, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, it was the chi-square that proved most useful. Conclusions: The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy.
Project description:Background: The analysis of oligonucleotide microarray data in pathogen surveillance and discovery assays is a challenging task. Target template concentration, nucleic acid integrity, and host nucleic acid composition can each have a profound effect on signal distribution. Exploratory analysis of fluorescent signal distribution in clinical samples has revealed deviations from normality, suggesting that distribution-free approaches should be applied. Results: An examination of both positive predictive value and false positive rates was employed to assess the utility of three well-established nonparametric methods for the analysis of viral array hybridization data: (1) Mann-Whitney U, (2) the Spearman correlation coefficient and (3) the chi-square test. Of the three tests, it was the chi-square that proved most useful. Conclusions: The acceptance of microarray use for routine clinical diagnostics will require that the technology be accompanied by simple yet reliable analytic methods. We report that our implementation of the chi-square test yielded a combination of low false positive rates and a high degree of predictive accuracy. A set of comprehensive probes covering vertebrate viruses was designed and printed using Agilent in-situ fabrication. Cells in tissue culture were infected with various viruses, then RNA was harvested. RNA was converted to cDNA, then amplified, labeled and hybridized to the array.
Project description:Fur seal feces-associated circular ssDNA virus (FSfaCV) was discovered in a pig for the first time in Japan using a next-generation sequencer with duplex-specific nuclease. Full genome of the virus showed approximately 92% similarity to FSfaCVs from New Zealand fur seals. Furthermore, we investigated the prevalence of the ssDNA virus in 85 piglets in Japan, and 65 piglets were positive (76%) for the virus.
Project description:During routine electron microscopy of fecal samples from diarrheic dogs dated from 2000 virus particles resembling circovirus in shape and size were detected in two samples (V2177/00; V3374/00). Polymerase chain reaction (PCR) using primers specific for porcine circovirus type 2 (PCV2) amplified DNA recovered from both samples. Sequencing of PCR amplificates (V2177/00) obtained with PCV2-specific primer pairs revealed a genome size of 1768bp. The nucleotide sequence was highly similar (98% nucleotide identity) to the PCV2a reference sequence.
Project description:Next-Generation-Sequencing (NGS) technologies have led to important improvement in the detection of new or unrecognized infective agents, related to infectious diseases. In this context, NGS high-throughput technology can be used to achieve a comprehensive and unbiased sequencing of the nucleic acids present in a clinical sample (i.e. tissues). Metagenomic shotgun sequencing has emerged as powerful high-throughput approaches to analyze and survey microbial composition in the field of infectious diseases. By directly sequencing millions of nucleic acid molecules in a sample and matching the sequences to those available in databases, pathogens of an infectious disease can be inferred. Despite the large amount of metagenomic shotgun data produced, there is a lack of a comprehensive and easy-use pipeline for data analysis that avoid annoying and complicated bioinformatics steps. Here we present HOME-BIO, a modular and exhaustive pipeline for analysis of biological entity estimation, specific designed for shotgun sequenced clinical samples. HOME-BIO analysis provides comprehensive taxonomy classification by querying different source database and carry out main steps in metagenomic investigation. HOME-BIO is a powerful tool in the hand of biologist without computational experience, which are focused on metagenomic analysis. Its easy-to-use intrinsic characteristic allows users to simply import raw sequenced reads file and obtain taxonomy profile of their samples.