Shale Dames

Next-Generation Sequencing: From Basic Research to Diagnostics

BACKGROUND For the past 30 years, the Sanger method has been the dominant approach and gold standard for DNA sequencing. The commercial launch of the first massively parallel pyrosequencing platform in 2005 ushered in the new era of high-throughput genomic analysis now referred to as next-generation sequencing (NGS). CONTENT This review describes fundamental principles of commercially available NGS platforms. Although the platforms differ in their engineering configurations and sequencing chemistries, they share a technical paradigm in that sequencing of spatially separated, clonally amplified DNA templates or single DNA molecules is performed in a flow cell in a massively parallel manner. Through iterative cycles of polymerase-mediated nucleotide extensions or, in one approach, through successive oligonucleotide ligations, sequence outputs in the range of hundreds of megabases to gigabases are now obtained routinely. Highlighted in this review are the impact of NGS on basic research, bioinformatics considerations, and translation of this technology into clinical diagnostics. Also presented is a view into future technologies, including real-time single-molecule DNA sequencing and nanopore-based sequencing. SUMMARY In the relatively short time frame since 2005, NGS has fundamentally altered genomics research and allowed investigators to conduct experiments that were previously not technically feasible or affordable. The various technologies that constitute this new paradigm continue to evolve, and further improvements in technology robustness and process streamlining will pave the path for translation into clinical diagnostics.

Next Generation Sequencing for Clinical Diagnostics-Principles and Application to Targeted Resequencing for Hypertrophic Cardiomyopathy: A Paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology

During the past five years, new high-throughput DNA sequencing technologies have emerged; these technologies are collectively referred to as next generation sequencing (NGS). By virtue of sequencing clonally amplified DNA templates or single DNA molecules in a massively parallel fashion in a flow cell, NGS provides both qualitative and quantitative sequence data. This combination of information has made NGS the technology of choice for complex genetic analyses that were previously either technically infeasible or cost prohibitive. As a result, NGS has had a fundamental and broad impact on many facets of biomedical research. In contrast, the dissemination of NGS into the clinical diagnostic realm is in its early stages. Though NGS is powerful and can be envisioned to have multiple applications in clinical diagnostics, the technology is currently complex. Successful adoption of NGS into the clinical laboratory will require expertise in both molecular biology techniques and bioinformatics. The current report presents principles that underlie NGS including sequencing library preparation, sequencing chemistries, and an introduction to NGS data analysis. These concepts are subsequently further illustrated by showing representative results from a case study using NGS for targeted resequencing of genes implicated in hypertrophic cardiomyopathy.

Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities.

Rapid isothermal nucleic acid amplification technologies can enable diagnosis of human pathogens and genetic variations in a simple, inexpensive, user-friendly format. The isothermal exponential amplification reaction (EXPAR) efficiently amplifies short oligonucleotides called triggers in less than 10 min by means of thermostable polymerase and nicking endonuclease activities. We recently demonstrated that this reaction can be coupled with upstream generation of trigger oligonucleotides from a genomic target sequence, and with downstream visual detection using DNA-functionalized gold nanospheres. The utility of EXPAR in clinical diagnostics is, however, limited by a nonspecific background amplification phenomenon, which is further investigated in this report. We found that nonspecific background amplification includes an early phase and a late phase. Observations related to late phase background amplification are in general agreement with literature reports of ab initio DNA synthesis. Early phase background amplification, which limits the sensitivity of EXPAR, differs however from previous reports of nonspecific DNA synthesis. It is observable in the presence of single-stranded oligonucleotides following the EXPAR template design rules and generates the trigger sequence expected for the EXPAR template present in the reaction. It appears to require interaction between the DNA polymerase and the single-stranded EXPAR template. Early phase background amplification can be suppressed or eliminated by physically separating the template and polymerase until the final reaction temperature has been reached, thereby enabling detection of attomolar starting trigger concentrations.

Closed-tube SNP genotyping without labeled probes: A comparison between unlabeled probe and amplicon melting

Two methods for closed-tube single nucleotide polymorphism (SNP) genotyping without labeled probes have become available: unlabeled probe and amplicon melting. Unlabeled probe and amplicon melting assays were compared using 5 SNPs: human platelet antigens 1, 2, 5, and 15 and a C>T variant located 13910 base pairs (bp) upstream of the lactase gene. LCGreen Plus (Idaho Technology, Salt Lake City, UT) was used as the saturating DNA dye. Unlabeled probe data were readily interpretable and accurate for all amplicon lengths tested. Five targets that ranged in size from 42 to 72 bp were well resolved by amplicon melting on the LightScanner (Idaho Technology) or LightTyper (Roche, Indianapolis, IN) with no errors in genotyping. However, when larger amplicons (206 bp) were used and analyzed on lower resolution instruments (LightTyper and I-Cycler, Bio-Rad, Hercules, CA), the accuracy of amplicon genotyping was only 73% to 77%. When 2 temperature standards were used to bracket the amplicon of interest, the accuracy of amplicon genotyping of SNPs was increased to 100% (LightTyper) and 88% (I-Cycler).

The Development of Next-Generation Sequencing Assays for the Mitochondrial Genome and 108 Nuclear Genes Associated with Mitochondrial Disorders

Sanger sequencing of multigenic disorders can be technically challenging, time consuming, and prohibitively expensive. High-throughput next-generation sequencing (NGS) can provide a cost-effective method for sequencing targeted genes associated with multigenic disorders. We have developed a NGS clinical targeted gene assay for the mitochondrial genome and for 108 selected nuclear genes associated with mitochondrial disorders. Mitochondrial disorders have a reported incidence of 1 in 5000 live births, encompass a broad range of phenotypes, and are attributed to mutations in the mitochondrial and nuclear genomes. Approximately 20% of mitochondrial disorders result from mutations in mtDNA, with the remaining 80% found in nuclear genes that affect mtDNA levels or mitochondrion protein assembly. In our NGS approach, the 16,569-bp mtDNA is enriched by long-range PCR and the 108 nuclear genes (which represent 1301 amplicons and 680 kb) are enriched by RainDance emulsion PCR. Sequencing is performed on Illumina HiSeq 2000 or MiSeq platforms, and bioinformatics analysis is performed using commercial and in-house developed bioinformatics pipelines. A total of 16 validation and 13 clinical samples were examined. All previously reported variants associated with mitochondrial disorders were found in validation samples, and 5 of the 13 clinical samples were found to have mutations associated with mitochondrial disorders in either the mitochondrial genome or the 108 nuclear genes. All variants were confirmed by Sanger sequencing.

Molecular Pathology Methods

Molecular pathology is based on the principles, techniques, and tools of molecular biology as they are applied to diagnostic medicine in the clinical laboratory. These tools were developed in the research setting and perfected throughout the second half of the 20th century, long before the Human Genome Project was conceived. Molecular biology methods were used to elucidate the genetic and molecular basis of many diseases, and these discoveries ultimately led to the field of molecular diagnostics. Eventually the insights these tools provided for laboratory medicine were so valuable to the armamentarium of the pathologist that they were incorporated into pathology practice. Today, molecular diagnostics continues to grow rapidly as in vitro diagnostic companies develop new kits for the marketplace and as the insights into disease gained by the progress of the Human Genome Project develop into laboratory tests.

A High-Throughput Next-Generation Sequencing Assay for the Mitochondrial Genome

Next-generation sequencing (NGS) is an effective method for mitochondrial genome (mtDNA) sequencing and heteroplasmy detection. The following protocol describes an mtDNA enrichment method up to library preparation and sequencing on Illumina NGS platforms. A short command line alignment script is available for download via FTP.

A rapid gene sequencing panel strategy to facilitate precision neonatal medicine

Next-generation sequencing (NGS) has the potential of revolutionizing neonatal intensive care by providing early molecular diagnosis in infants with congenital disorders (Saunders et al., 2012; Soden et al., 2014;Willig et al., 2015). These efforts have thus far largely employed rapid whole-exome (WES) and whole-genome sequencing (WGS), showing their utility in facilitating care in complex cases. Yet, a lack of practical selection criteria and the high costs of these tests have impeded the broad implementation of NGS into neonatal intensive care. We found that daily variable costs (those above “lights-on” expenses) in our level IV neonatal intensive care unit (NICU) at Primary Children’s Hospital are

BCAP31 Mutation Causing a Syndrome of Congenital Dystonia, Facial Dysorphism and Central Hypomyelination Discovered Using Exome Sequencing

2,100–3,300 (median to 75th centile), although the cost to care for critically ill neonates can be far more in the 1st weeks of life.We also observed a pattern of time-consuming and expensive diagnostic odysseys in infants with undiagnosed congenital disorders, sometimes culminating in a diagnostic exome at several months of age. We therefore hypothesized that early use of a rapid large NGS panel might have the highest clinical utility in these infants. Amultidisciplinary team at Primary Children’s Hospital and the University of Utah School of Medicine collaborated with Associated Regional and University Pathologists, Inc. (ARUP) Laboratories to implement a practical, rapidNGS strategy. The team developed a panel of 4,503 known disease-associated genes (Table 1 and Supplemental Table S1) in an attempt to maximize clinical utility while minimizing cost. NICU-specific patient selection criteria were defined (Table 2). The panel became available in the fall of 2015 at a cost per patientmother-father trio of

Gene silencing in Caenorhabditis elegans by transitive RNA interference

6,000 and a 7–10 day preliminary turnaround time. Of the twelve patients tested to date, we identified a diseasecausing variant in eight cases (67%), providing a plausible explanation for the phenotypes (Table 3). Six of these cases resulted from de novo pathogenic variants, one was due to a maternally inherited pathogenic variant in a paternally imprinted gene, and one was due to bi-parental inheritance in an autosomal recessive disorder. The latterwas an infant who presented at birth with profound hypotonia, diminished brainstem reflexes and ventilator dependency of unknown etiology. We identified two pathogenic variants in the CHAT gene consistent with congenital myasthenic syndrome, a condition often requiring a lengthy diagnostic workup. Anticholinesterases were administered within 48 hr of the preliminary report, leading to improved motor function. Therapy titration facilitated discharge to an intermediate care facility by 65 days of age. Overall, preliminary and final results had no discrepancy and were received after a mean of 9 and 16 days, respectively. In none of the eight positive cases the diagnosis was strongly suspected prior to the rapid gene panel. Throughout this study our approach has been to send more specific genetic testing in case there was a strong suspicion for a unique disease. Importantly, these data suggest that in the absence of one or both parent patients would have an uncertain diagnosis orwould be undiagnosed. There are a number of reasons for the relatively small number of patients analyzed over the described period. During the first year of the study the criteria for enrollment required patients to be in cardiac or respiratory failure. The guidelines were later widened to include patients withmultiple congenital anomalies (Table 2). The requirement of a trio presented additional challenges for enrollment. Importantly, in Current address: University of Nebraska and Children’s Hospital Medical Center, Department of Pediatrics (Neonatology), 8200 Dodge Street, Omaha NE 68124, Phone: 402-955-6140, Email: luca.brunelli@unmc.edu or lbrunelli@childrensomaha.org