Comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood.

BACKGROUND: The introduction of benchtop sequencers has made adoption of whole genome sequencing possible for a broader community of researchers than ever before. Concurrently, metagenomic sequencing (MGS) is rapidly emerging as a tool for interrogating complex samples that defy conventional analyses. In addition, next-generation sequencers are increasingly being used in clinical or related settings, for instance to track outbreaks. However, information regarding the analytical sensitivity or limit of detection (LoD) of benchtop sequencers is currently lacking. Furthermore, the specificity of sequence information at or near the LoD is unknown. RESULTS: In the present study, we assess the ability of three next-generation sequencing platforms to identify a pathogen (viral or bacterial) present in low titers in a clinically relevant sample (blood). Our results indicate that the Roche-454 Titanium platform is capable of detecting Dengue virus at titers as low as 1X102.5 pfu/mL, corresponding to an estimated 5.4X104 genome copies/ml maximum. The increased throughput of the benchtop sequencers, the Ion Torrent PGM and Illumina MiSeq platforms, enabled detection of viral genomes at concentrations as low as 1X104 genome copies/mL. Platform-specific biases were evident in sequence read distributions as well as viral genome coverage. For bacterial samples, only the MiSeq platform was able to provide sequencing reads that could be unambiguously classified as originating from Bacillus anthracis. CONCLUSION: The analytical sensitivity of all three platforms approaches that of standard qPCR assays. Although all platforms were able to detect pathogens at the levels tested, there were several noteworthy differences. The Roche-454 Titanium platform produced consistently longer reads, even when compared with the latest chemistry updates for the PGM platform. The MiSeq platform produced consistently greater depth and breadth of coverage, while the Ion Torrent was unequaled for speed of sequencing. None of the platforms were able to verify a single nucleotide polymorphism responsible for antiviral resistance in an Influenza A strain isolated from the 2009 H1N1 pandemic. Overall, the benchtop platforms perform well for identification of pathogens from a representative clinical sample. However, unlike identification, characterization of pathogens is likely to require higher titers, multiple libraries and/or multiple sequencing runs.

Relation between atherosclerosis risk factors and aspirin resistance in a primary prevention population.

Resistance to inhibition of platelet function by aspirin may contribute to future myocardial infarction and stroke. Adverse cardiovascular outcomes have been associated with aspirin resistance on several different platelet function assays, including the level of urinary 11-dehydro thromboxane B2 (Tx-M), platelet aggregation to arachidonic acid and adenosine diphosphate, and closure time on the platelet function analyzer-100. We examined the concordance of these aspirin-resistance assays and their relation to cardiovascular risk factors in a primary prevention population. Asymptomatic patients (n = 1,311) at increased risk for coronary heart disease were evaluated before and after 2 weeks of aspirin (81 mg/day). Aspirin resistance was defined according to published criteria for these 3 assays of platelet function. Subjects were characterized for the presence of atherosclerosis risk factors. Agreement among the 3 assays was poor. Only 5 patients met aggregation criteria for aspirin resistance. Attenuated suppression of urinary Tx-M by aspirin was associated with a greater atherosclerotic risk profile and Framingham risk score in multivariable regression analysis. Aspirin resistance by platelet function analyzer-100 was associated only with increased von Willebrand factor levels and not with atherosclerotic risk profile. In conclusion, in a primary prevention population, different published criteria for aspirin resistance classify distinct groups of patients as aspirin resistant with very little overlap. Higher Tx-M, which reflects decreased suppression of thromboxane production in vivo, is the only criterion associated with atherosclerosis risk factors, suggesting that this measurement may represent the most relevant approach for identifying asymptomatic subjects whose aspirin treatment will "fail."

BLASTPLOT: a PERL module to plot next generation sequencing NCBI-BLAST results.

BACKGROUND: The development of Next Generation Sequencing (NGS) during the last decade has created an unprecedented amount of sequencing data, as well as the ability to rapidly sequence specimens of interest. Read-based BLAST analysis of NGS data is a common procedure especially in the case of metagenomic samples. However, coverage is usually not enough to allow for de novo assembly. This type of read-based analysis often creates the question of how the reads that align to the same sequence are distributed. The same question applies to preparation of primers or probes for microarray experiments. Although there are several packages that allow the visualization of DNA segments in relation to a reference, in most cases they require the visualization of one reference at a time and the capture of screen shots for each segment. Such a procedure could be tedious and time consuming. The field is in need of a solution that automates the capture of coverage plots for all the segments of interest. RESULTS: We have created BLASTPLOT, a PERL module to quickly plot the BLAST results from short sequences (primers, probes, reads) against reference targets. CONCLUSIONS: BLASTPLOT is a simple to use PERL module that allows the generation of PNG graphs for all the reference sequences associated with a BLAST result set.

OGA: an ontological tool of human phenotypes with genetic associations.

BACKGROUND: The availability of genetic data has increased dramatically in recent years. The greatest value of this data is its potential for personalized medicine. Many new associations are reported every day from Genome Wide Association Studies (GWAS). However, robust, reproducible associations are elusive for some complex diseases. Ontologies present a potential way to distinguish between spurious associations and those with a potential influence on the phenotype. Such an approach would be based on finding associations of the same genetic variant with closely related, but distinct, phenotypes. This approach can be accomplished with a phenotype ontology that also holds genetic association data. RESULTS: Here, we report a structured knowledge application to navigate and to facilitate the discovery of relationships between different phenotypes and their genetic associations. CONCLUSIONS: OGA allows users to (1) find the intersecting set of genes for phenotypes of interest, (2) find empirical p values for such observations and (3) OGA outperforms similar applications in number of total concepts and genes mapped.