Improving the Diagnosis and Management of Serious Infection Using Nanopore Metagenomic Sequencing
University of East Anglia, United Kingdom
Rapid and accurate diagnosis is critical for the effective treatment of life threatening infections, such as bloodstream, respiratory tract and complicated urinary tract infections. These clinical syndromes have complex aetiology and require the recognition of pathogens within challenging sample matrices. The “gold standard” culture techniques are labour intensive, have long turn-around times (≥2 days) and often offer poor clinical sensitivity. In the absence of rapidly available pathogen or resistance information, the patient is treated empirically with broad spectrum therapy. This empirical therapy is often unnecessarily potent or, conversely, sometimes ineffective due to inherent or acquired resistance.
A paradigm shift in diagnostics technology is required, to allow the development of a universal diagnostic which can detect any pathogen or resistance. Shotgun metagenomics sequencing has the potential to drive this shift by combining rapidity with comprehensiveness beyond that of culture or PCR. Nanopore real-time sequencing technology has, for the first time, made it feasible to apply metagenomic sequencing to acute infection diagnosis.
By providing rapid pathogen identification and AMR profiling, metagenomic sequencing will afford clinicians with a timely and comprehensive diagnosis, reducing empiric treatment to a single dose and enabling tailored antimicrobial therapy. I will discuss our ongoing research on the development of metagenomic sequencing based diagnostic tests for bloodstream, urinary tract and respiratory tract infections.
Applications of an in vitro Experimental Model for a Systems Level Understanding of Health and Disease States of the Human Oral Microbiome
1J Craig Venter Institute, Genome Medicine Group, CA, USA; 2University of Central Florida, College of Engineering and Computer Science, FL, USA; 3University of California Los Angeles, Shool of Dentistry, CA, USA; 4University of Washington, School of Dentistry, WA, USA
Although oral microbial communities are subjected to daily physical and chemical disturbances such as fluctuations in pH, antimicrobial agents, dietary components and personal hygiene measures, a long-term stable microbiome persists. Biological processes that support this stability are important for the prevention of dysbiosis—a microbial shift toward a disease, e.g. periodontitis (gum disease) or caries (tooth decay), the two most common infectious diseases of man. In our previous work, by studying an oral in vitro biofilm model system greater than 100 bacterial species, we identified a plethora of metabolic activities possibly associated with oral health both at the gene and molecule level. We also showed that metabolic activities varied greatly for individual bacterial key-community members during pH fluctuations. Here, we focus on understanding molecular mechanisms and species interactions critical for biofilm community stability during a 24-hour incubation period. By applying meta-omics approaches we dissect regulatory pathways that control the plankton-to-biofilm transition and the maintenance of the stable oral biofilm community during pH stress. These approaches also allow us to target gene transcription activities of virulence mechanisms in low pH over time.
A Novel Approach for Selective Enrichment of Custom Gene Targets for Oncology Research
1New England Biolabs GmbH, Germany; 2New England Biolabs Inc., U.S.; 3Directed Genomics Inc., U.S.
Target enrichment of selected exonic regions for deep sequence analysis is a widely used practice for the discovery of novel variants, identification and phenotypic association of known variants for a wide range of practical applications, including somatic variant detection in human cancer.
Deep sequencing of tumor material is required to effectively detect mutations that may be present at low frequencies, or in samples that contain a mixture of malignant and stromal cells. Due to these challenges, highly focused gene panels are used to narrow the regions being studied, reducing overall sequencing costs while providing the necessary coverage for variant detection. Challenges pertaining to selecting the appropriate genes for inclusion are further confounded by the high costs and time required to develop custom gene panels.
The NEBNext DirectTM technology utilizes a novel approach to selectively enrich nucleic acid targets ranging from a single gene to several hundred genes, without sacrificing specificity. Furthermore, intrinsic properties of the approach improve sensitivity and have proven amenable to challenging sample types including FFPE tissue and circulating tumor DNA (ctDNA). The result is a 1-day protocol that enables the preparation of sequence-ready libraries with high specificity, uniformity, and sensitivity for the discovery and identification of nucleic acid variants.
Here, we will discuss the NEBNext Direct approach to target enrichment, specifically with regard to identification of somatic variants in clinically relevant samples, as well as content strategies to efficiently customize content for genes involved in cancer research.
Liquid biopsies, biomarker signatures, and beyond - why standardization of small RNA-Seq matters
1Department of Animal Physiology and Immunology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany; 2Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Germany; 3Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University, Munich, Germany; 4Department of Physiology, TUM School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany; 5Eurofins Medigenomix Forensik GmbH, Ebersberg, Germany
Small RNA-Seq has revolutionized transcriptomics research in much the same way RT-qPCR did several decades ago. The massively parallel sequencing of short RNA reads has already yielded unprecedented insights in areas such as gene expression profiling, clinical diagnostics, and biomarker discovery. Researchers are simultaneously intrigued by the technology’s promises, and challenged with a multitude of hurdles on the way to accurate and meaningful data derived from high-throughput sequencing. Factors impacting experimental outcomes range from wet lab parameters such as sample type and quality, RNA extraction, and library preparation chemistry to processing and analysing massive amounts of data in the post-experimental phase. In addition to the inherent complexity brought about by multiple types of small RNA being studied in various biological scenarios, the choice from a plethora of commercially available sample processing kits, absence of comprehensive guidelines, and suboptimal reporting further complicate the body of literature. Reliable and reproducible research findings can only be realized by rigorous experimental standardization and validation complemented with extensive and transparent reporting of procedures in small RNA-Seq manuscripts. Forming hypotheses based on assumptions developed from flawed data leads to inconsistent findings across the literature and impedes translation of scientific discoveries into much-anticipated clinical applications such as molecular diagnostics and development of liquid biopsy-based disease biomarkers. Even though the parallels between RT-qPCR and RNA-Seq for nucleic acid quantification are obvious, it could be argued that Next-Generation Sequencing techniques bear even more risk for biases due to the complexity of post-experimental data processing. The widely-adopted MIQE guidelines clearly demonstrate how authoritative guidelines and quality standards translate into improved reporting, better experimental setups and, ultimately, valuable applications of research findings. Focusing on the development of biomarker signatures, we herein similarly point out challenges along the small RNA-Seq workflow, report common sources of experimental bias, and call for rigorous quality control and validation in order to generate high-quality, reproducible and meaningful sequencing data.
Single-tube library prep solutions for high quality DNA sequencing
1Quantabio, United States of America; 2QIAGEN Beverly, United States of America
With wide adoption of NGS in research and healthcare, simple, rapid, and reliable solutions for high quality NGS library preparation are in great demand as library preparation can greatly impact the sequencing results and overall sample to sequencing cost. To offer high-quality and streamlined library preparation solutions for the Illumina® platform, we have developed two solutions for different sequencing library preparations. The first solution combines DNA fragmentation and library preparation in a single tube. This provides easily tunable enzymatic fragmentation, end-repair and dA-tailing in a single step, followed by high efficiency adapter ligation in the same reaction tube without intervening purification steps. The novel chemistry and simplified workflow lead to DNA libraries with high complexity and even GC coverage uniformity. For sample types which do not require DNA fragmentation, our second library prep product provides a fast, single tube library preparation solution ensuring enhanced sensitivity and efficiency, while maximizing library yields with minimal hands-on time. Leveraging our innovative chemistries, world-class enzyme purity and rigorously controlled production and quality system, both single tube solutions offer unmatchable library prep results that address quality, speed, and throughput while remaining a cost-effective option.
Forensic Application Of Microbiome Profiling
1Eurofins Medigenomix GmbH, Germany; 2Eurofins Medigenomix Forensik GmbH, Germany
The microbiome is defined as the collective genomes of the microbes that live inside and on the human body. In a forensic context, this is the collective genomes of the microbes that are present in a stain sample. Investigating such samples with human specific STR-markers ignores the information that might be present in this part of the extracted DNA. A forensic application could either use this for (additional) identification of the stain donor or for the investigation of the source level of a stain. We investigated the microbiomes of skin, mouth, nose and vaginal swabs from a set of volunteers by V4 16S ribosomal RNA massive parallel sequencing. Results of this study show the great diversity of the microflora. The species observed by us in the various sample types overlap to some extent. Potential applications of the data are discussed.
Challenges of targeted NGS analyses
ecSeq GmbH, Germany
Breast cancer is one of the most distributed and investigated cancer types available and germline mutations in the BRCA1/2 genes are known to lead to a high lifetime probability of developing breast cancer. The application of targeted next-generation sequencing (NGS) to this cancer type has enabled the development of cost-effective and rapid tools for diagnostics and treatment response/resistance. Even though available bioinformatics analysis tools for detecting mutations in BRCA1/2 are highly developed and popular, there are still issues with incorrectly called variants. This talk will cover some of these problems and suggest solutions for targeted NGS data analysis.