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Slezak, Tom

Publications and source records attributed to Slezak, Tom.

5 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2019

FDA-ARGOS is a database with public quality-controlled reference genomes for diagnostic use and regulatory science

FDA proactively invests in tools to support innovation of emerging technologies, such as infectious disease next generation sequencing (ID-NGS). Here, we introduce FDA-ARGOS quality-controlled reference genomes as a public database for diagnostic purposes and demonstrate its utility on the example of two use cases. We provide quality control metrics for the FDA-ARGOS genomic database resource and outline the need for genome quality gap filling in the public domain. In the first use case, we show more accurate microbial identification of Enterococcus avium from metagenomic samples with FDA-ARGOS reference genomes compared to non-curated GenBank genomes. In the second use case, we demonstrate the utility of FDA-ARGOS reference genomes for Ebola virus target sequence comparison as part of a composite validation strategy for ID-NGS diagnostic tests. The use of FDA-ARGOS as an in silico target sequence comparator tool combined with representative clinical testing could reduce the burden for completing ID-NGS clinical trials

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Axiom Microbiome Array, the next generation microarray for high-throughput pathogen and microbiome analysis

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been utilized to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. In this study, the Axiom Microbiome Array was evaluated to discover its sensitivity, specificity and utility in microbiome analysis of veterinary clinical samples. The array contains probes designed to detect more than 12,000 species of viruses, bacteria, fungi, protozoa and archaea, yielding the most comprehensive microbial detection platform built to date. The array was able to detect Shigella and Aspergillus at 100 genome copies, and vaccinia virus DNA at 1,000 genome copies. The Axiom Microbiome Array made correct species-level calls in mock microbial community samples. When tested against serum, tissue, and fecal samples from pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, the microarray correctly detected these two viruses and other common viral and bacterial microbiome species. This cost-effective and high-throughput microarray is an efficient tool to rapidly analyze large numbers of clinical and environmental samples for the presence of multiple viral and bacterial pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Simulate_PCR for amplicon prediction and annotation from multiplex, degenerate primers and probes

Background: Pairing up primers to amplify desired targets and avoid undesired cross reactions can be a combinatorial challenge. Effective prediction of specificity and inclusivity from multiplexed primers and TaqMan®/ Luminex® probes is a critical step in PCR design. Results: Code is described to identify all primer and probe combinations from a list of unpaired, unordered candidates that should produce a product. It predicts and extracts all amplicon sequences in a large sequence database from a list of primers and probes, allowing degenerate bases and user-specified levels of primer-target mismatch tolerance. Amplicons hit by TaqMan®/Luminex® probes are indicated, and products may be annotated with gene information from NCBI. Fragment length distributions are calculated to predict electrophoretic gel banding patterns. Conclusions: Simulate_PCR is the only freely available software that can be run from the command line for high throughput applications which can calculate all products from large lists of primers and probes compared to a large sequence database such as nt. It requires no prior knowledge of how primers should be paired. Degenerate bases are allowed and entire amplicon sequences are extracted and annotated with gene information. Examples are provided for sets of TaqMan®/Luminex® PCR signatures predicted to amplify all HIV-1 genomes, all Coronaviridae genomes, and a group of antibiotic resistance genes. The software is a command line perl script freely available as open source.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Analysis of sensitivity and rapid hybridization of a multiplexed Microbial Detection Microarray

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. A multiplexed version of the Lawrence Livermore Microbial Detection Array (LLMDA) was developed and evaluated with minimum detectable concentrations for pure unamplified DNA viruses, along with mixtures of viral and bacterial DNA subjected to different whole genome amplification protocols. In addition the performance of the array was tested when hybridization time was reduced from 17 h to 1 h. The LLMDA was able to detect unamplified vaccinia virus DNA at a concentration of 14 fM, or 100,000 genome copies in 12 μL of sample. With amplification, positive identification was made with only 100 genome copies of input material. When tested against human stool samples from patients with acute gastroenteritis, the microarray detected common gastroenteritis viral and bacterial infections such as rotavirus and E. coli. Accurate detection was found but with a 4-fold drop in sensitivity for a 1 h compared to a 17 h hybridization. The array detected 2 ng (equivalent concentration of 15.6 fM) of labeled DNA from a virus with 1 h hybridization without any amplification, and was able to identify the components of a mixture of viruses and bacteria at species and in some cases strain level resolution. Sensitivity improved by three orders of magnitude with random whole genome amplification prior to hybridization; for instance, the array detected a DNA virus with only 20 fg or 100 genome copies as input. This multiplexed microarray is an efficient tool to analyze clinical and environmental samples for the presence of multiple viral and bacterial pathogens rapidly.

59 BASIC BIOLOGICAL SCIENCES↗