Clinical metagenomics.

Clinical metagenomic next-generation sequencing (mNGS), the comprehensive analysis of microbial and host genetic material (DNA and RNA) in samples from patients, is rapidly moving from research to clinical laboratories. This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology. Here, we focus on the challenges of implementing mNGS in the clinical laboratory and address potential solutions for maximizing its impact on patient care and public health

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries

Genome targeting methods enable cost-effective capture of specific subsets of the genome for sequencing. We present here an automated, highly scalable method for carrying out the Solution Hybrid Selection capture approach that provides a dramatic increase in scale and throughput of sequence-ready libraries produced. Significant process improvements and a series of in-process quality control checkpoints are also added. These process improvements can also be used in a manual version of the protocol

Clinical application of high throughput molecular screening techniques for pharmacogenomics.

Genetic analysis is one of the fastest-growing areas of clinical diagnostics. Fortunately, as our knowledge of clinically relevant genetic variants rapidly expands, so does our ability to detect these variants in patient samples. Increasing demand for genetic information may necessitate the use of high throughput diagnostic methods as part of clinically validated testing. Here we provide a general overview of our current and near-future abilities to perform large-scale genetic testing in the clinical laboratory. First we review in detail molecular methods used for high throughput mutation detection, including techniques able to monitor thousands of genetic variants for a single patient or to genotype a single genetic variant for thousands of patients simultaneously. These methods are analyzed in the context of pharmacogenomic testing in the clinical laboratories, with a focus on tests that are currently validated as well as those that hold strong promise for widespread clinical application in the near future. We further discuss the unique economic and clinical challenges posed by pharmacogenomic markers. Our ability to detect genetic variants frequently outstrips our ability to accurately interpret them in a clinical context, carrying implications both for test development and introduction into patient management algorithms. These complexities must be taken into account prior to the introduction of any pharmacogenomic biomarker into routine clinical testing

THE EFFECT OF STRUCTURE IN SHORT REGIONS OF DNA ON MEASUREMENTS ON SHORT OLIGONUCLEOTIDE MICROARRAY AND ION TORRENT PGM SEQUENCING PLATFORMS

Single-stranded DNA in solution has been studied by biophysicists for many years, as complex structures, both stable and dynamic, form under normal experimental conditions. Stable intra-strand formations affect enzymatic technical processes such as PCR and biological processes such as gene regulation. In the research described here we examined the effect of such structures on two high-throughput genomic assay platforms and whether we could predict the influence of those effects to improve the interpretation of genomic sequencing results. Helical structures in DNA can be composed of interactions across strands or within a strand. Exclusion of the aqueous solvent provides an entropic advantage to more compact structures. Our first experiments were tested whether internal helical regions in one of the two binding partners in a microarray experiment would influence the stability of the complex. Our results are novel and show, from molecular simulations and hybridization experiments, that stable secondary structures on the boundary, when not impinging on the ability of targets to access the probes, stabilize the probe-target hybridization. High-throughput sequencing (HTS) platforms use as templates short single-stranded DNA fragments. We tested the influence of template secondary structure on the fidelity of reads generated using the Ion Torrent PGM platform. It can clearly be seen for targets where hairpin structures are quite long (~20bp) that a high level of mis-calling occurs, particularly of deletions, and that some of these deletions are 20-30 bases long. These deletions are not associated with homopolymers, which are known to cause base mis-calls on the PGM, and the effect of structure on the sequencing reaction, rather than the PCR preparative steps, has not been previously published. As HTS technologies bring the cost of sequencing whole genomes down, a number of unexpected observations have arisen. An example that caught our attention is the prevalence of far more short deletions than had been detected using Sanger methods. The prevalence is particularly high in the Korean genome. Since we showed that helical structures could disrupt the fidelity of base calls on the Ion Torrent we looked at the context of the apparent deletions to determine whether any sequence or structure pattern discriminated them. Starting with the genome provided by Kim et al (1) we selected deletions > 2 bases long from chromosome I of a Korean genome. We created 70 nucleotide fragments centered on the deletion. We simulated the secondary structures using OMP software and then modeled using the Random Forest algorithm in the WEKA modeling package to characterize the relations between the deletions and secondary structures in or around them. After training the model on chromosome I deletions we tested it using chromosome 20 deletions. We show that sequence information alone is not able to predict whether a deletion will occur, while the addition of structural information improves the prediction rates. Classification rates are not yet high: additional data and a more precise structural description are likely needed to train a robust model. We are unable to state which of the structures affect in vitro platforms and which occur in vivo. A comparative genomics approach using 38 genomes recently made available for the CAMDA 2013 competition should provide the necessary information to train separate models if the important features are different in the two cases

Recent trends in molecular diagnostics of yeast infections : from PCR to NGS

The incidence of opportunistic yeast infections in humans has been increasing over recent years. These infections are difficult to treat and diagnose, in part due to the large number and broad diversity of species that can underlie the infection. In addition, resistance to one or several antifungal drugs in infecting strains is increasingly being reported, severely limiting therapeutic options and showcasing the need for rapid detection of the infecting agent and its drug susceptibility profile. Current methods for species and resistance identification lack satisfactory sensitivity and specificity, and often require prior culturing of the infecting agent, which delays diagnosis. Recently developed high-throughput technologies such as next generation sequencing or proteomics are opening completely new avenues for more sensitive, accurate and fast diagnosis of yeast pathogens. These approaches are the focus of intensive research, but translation into the clinics requires overcoming important challenges. In this review, we provide an overview of existing and recently emerged approaches that can be used in the identification of yeast pathogens and their drug resistance profiles. Throughout the text we highlight the advantages and disadvantages of each methodology and discuss the most promising developments in their path from bench to bedside

Capturing the ‘ome’ : the expanding molecular toolbox for RNA and DNA library construction

All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application