Comparative genomics tools applied to bioterrorism defence

Abstract

Rapid advances in the genomic sequencing of bacteria and viruses over the past few years have made it possible to consider sequencing the genomes of all pathogens that affect humans and the crops and livestock upon which our lives depend. Recent events make it imperative that full genome sequencing be accomplished as soon as possible for pathogens that could be used as weapons of mass destruction or disruption. This sequence information must be exploited to provide rapid and accurate diagnostics to identify pathogens and distinguish them from harmless near-neighbours and hoaxes. The Chem-Bio Non-Proliferation (CBNP) programme of the US Department of Energy (DOE) began a large-scale effort of pathogen detection in early 2000 when it was announced that the DOE would be providing bio-security at the 2002 Winter Olympic Games in Salt Lake City, Utah. Our team at the Lawrence Livermore National Lab (LLNL) was given the task of developing reliable and validated assays for a number of the most likely bioterrorist agents. The short timeline led us to devise a novel system that utilised whole-genome comparison methods to rapidly focus on parts of the pathogen genomes that had a high probability of being unique. Assays developed with this approach have been validated by the Centers for Disease Control (CDC). They were used at the 2002 Winter Olympics, have entered the public health system, and have been in continual use for non-publicised aspects of homeland defence since autumn 2001. Assays have been developed for all major threat list agents for which adequate genomic sequence is available, as well as for other pathogens requested by various government agencies. Collaborations with comparative genomics algorithm developers have enabled our LLNL team to make major advances in pathogen detection, since many of the existing tools simply did not scale well enough to be of practical use for this application. It is hoped that a discussion of a real-life practical application of comparative genomics algorithms may help spur algorithm developers to tackle some of the many remaining problems that need to be addressed. Solutions to these problems will advance a wide range of biological disciplines, only one of which is pathogen detection. For example, exploration in evolution and phylogenetics, annotating gene coding regions, predicting and understanding gene function and regulation, and untangling gene networks all rely on tools for aligning multiple sequences, detecting gene rearrangements and duplications, and visualising genomic data. Two key problems currently needing improved solutions are: (1) aligning incomplete, fragmentary sequence (eg draft genome contigs or arbitrary genome regions) with both complete genomes and other fragmentary sequences; and (2) ordering, aligning and visualising non-colinear gene rearrangements and inversions in addition to the colinear alignments handled by current tools.

Utilization of multiple “omics” studies in microbial pathogeny for microbiology insights

“…Apart from the already mentioned case of new virus in 2009, it can be applied in other situation. The best example is the comparative genomics tools applied to bioterrorism defence [15] .…”

Diagnostic Evaluation of Multiplexed Reverse Transcription-PCR Microsphere Array Assay for Detection of Foot-and-Mouth and Look-Alike Disease Viruses

“…Primer-probe sequences for FMDV (4,30) and BVDV (7,22) were based on the work of others and adapted to the current multiplex format. All other sequences were designed at the Lawrence Livermore National Laboratory using an approach that was described previously (36). Additional computational analyses were performed to ensure the specificity and reliability of all available data, including a BLAST-based comparison of each primer-probe set as a triplet against all sequences in GenBank to identify the targets that are predicted to produce a PCR or TaqMan reaction at 57°C for primer annealing and 67°C for probe annealing, where temperatures are derived from Primer 3 oligonucleotide melting point calculations.…”

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

“…Page 11 of 55 Laboratory (LLNL) using an approach that has previously been described (36 . Ŧ = an internal biotinylated dT.…”