Access To Next Generation Sequencing Research Articles

Treatment of Cholestasis in Infants and Young Children.

Cholestasis is characterized by a conjugated hyperbilirubinemia secondary to impaired bile synthesis, transport, or excretion from the liver. It is always pathologic and can be indicative of an underlying hepatobiliary, genetic, or metabolic disorder, several of which require timely diagnosis to ensure proper management and optimal outcomes. This review provides an overview of the evaluation of cholestasis with a focus on current and emerging treatment strategies. Increased accessibility of next generation sequencing (NGS) allows for utilization of genetic testing early in the diagnostic process. This may alter the clinical algorithm for diagnosis of cholestatic disorders. An enhanced understanding of the underlying pathophysiology may help guide future development of targeted therapies, such as ileal bile acid transporter (IBAT) inhibitors. These were recently approved for treatment of cholestatic pruritus in patients with Alagille syndrome and Progressive Familial Intrahepatic Cholestasis. Current management of cholestasis is aimed at the biochemical consequences of impaired bile flow, including malnutrition, pruritus, and progressive fibrosis. NGS has led to an enhanced understanding of biliary pathology and may guide development of future treatment modalities based on specific gene mutations. Rapid discernment of the underlying etiology is essential as new treatment modalities emerge.

Evolutionary genomics: Insights from the invasive European starlings.

Two fundamental questions for evolutionary studies are the speed at which evolution occurs, and the way that this evolution may present itself within an organism's genome. Evolutionary studies on invasive populations are poised to tackle some of these pressing questions, including understanding the mechanisms behind rapid adaptation, and how it facilitates population persistence within a novel environment. Investigation of these questions are assisted through recent developments in experimental, sequencing, and analytical protocols; in particular, the growing accessibility of next generation sequencing has enabled a broader range of taxa to be characterised. In this perspective, we discuss recent genetic findings within the invasive European starlings in Australia, and outline some critical next steps within this research system. Further, we use discoveries within this study system to guide discussion of pressing future research directions more generally within the fields of population and evolutionary genetics, including the use of historic specimens, phenotypic data, non-SNP genetic variants (e.g., structural variants), and pan-genomes. In particular, we emphasise the need for exploratory genomics studies across a range of invasive taxa so we can begin understanding broad mechanisms that underpin rapid adaptation in these systems. Understanding how genetic diversity arises and is maintained in a population, and how this contributes to adaptability, requires a deep understanding of how evolution functions at the molecular level, and is of fundamental importance for the future studies and preservation of biodiversity across the globe.

Using gene panels in the diagnosis of neuromuscular disorders: A mini-review.

The diagnosis of inherited neuromuscular disorders is challenging due to their genetic and phenotypic variability. Traditionally, neurophysiology and histopathology were primarily used in the initial diagnostic approach to these conditions. Sanger sequencing for molecular diagnosis was less frequently utilized as its application was a time-consuming and cost-intensive process. The advent and accessibility of next-generation sequencing (NGS) has revolutionized the evaluation process of genetically heterogenous neuromuscular disorders. Current NGS diagnostic testing approaches include gene panels, whole exome sequencing (WES), and whole genome sequencing (WGS). Gene panels are often the most widely used, being more accessible due to availability and affordability. In this mini-review, we describe the benefits and risks of clinical genetic testing. We also discuss the utility, benefits, challenges, and limitations of using gene panels in the evaluation of neuromuscular disorders.

A critical evaluation of Mycobacterium bovis pangenomics, with reference to its utility in outbreak investigation.

The increased accessibility of next generation sequencing has allowed enough genomes from a given bacterial species to be sequenced to describe the distribution of genes in the pangenome, without limiting analyses to genes present in reference strains. Although some taxa have thousands of whole genome sequences available on public databases, most genomes were sequenced with short read technology, resulting in incomplete assemblies. Studying pangenomes could lead to important insights into adaptation, pathogenicity, or molecular epidemiology, however given the known information loss inherent in analyzing contig-level assemblies, these inferences may be biased or inaccurate. In this study we describe the pangenome of a clonally evolving pathogen, Mycobacterium bovis , and examine the utility of gene content variation in M. bovis outbreak investigation. We constructed the M. bovis pangenome using 1463 de novo assembled genomes. We tested the assumption of strict clonal evolution by studying evidence of recombination in core genes and analyzing the distribution of accessory genes among core monophyletic groups. To determine if gene content variation could be utilized in outbreak investigation, we carefully examined accessory genes detected in a well described M. bovis outbreak in Minnesota. We found significant errors in accessory gene classification. After accounting for these errors, we show that M. bovis has a much smaller accessory genome than previously described and provide evidence supporting ongoing clonal evolution and a closed pangenome, with little gene content variation generated over outbreaks. We also identified frameshift mutations in multiple genes, including a mutation in glpK, which has recently been associated with antibiotic tolerance in Mycobacterium tuberculosis . A pangenomic approach enables a more comprehensive analysis of genome dynamics than is possible with reference-based approaches; however, without critical evaluation of accessory gene content, inferences of transmission patterns employing these loci could be misguided.

TBIO-12. Screening for Cancer Predisposition Syndromes in Pediatric Neuro-Oncology Patients: a Single Institution Experience

BACKGROUND: Traditional screening for cancer predisposition syndromes centers on family history, phenotypic features, and tumor histology. With expanded accessibility of next generation sequencing, identification of de novo germline mutations is increasing and the predictive value of family history has become less clear. While identification of germline mutations often affects care of pediatric neuro-oncology patients, germline sequencing is not currently standard of care. We hypothesize that routine screening for germline mutations in pediatric neuro-oncology patients reveals unsuspected cancer predisposition syndromes and impacts care. METHODS: A retrospective analysis was performed on pediatric neuro-oncology patients at a single institution who had targeted next generation sequencing of approximately 500-cancer associated genes (UCSF500) on tumor and paired germline DNA. We determined the proportion of patients with germline mutations and assessed impact on future screening and current tumor treatment. We interrogated clinical notes, family history, and interviewed treating physicians to determine if predispositions were previously suspected. RESULTS: Between June 2015-December 2019, 187 patients had paired testing; of these 29 (16%) harbored germlines mutations that were pathogenic or likely pathogenic. Germline mutations were found in patients with high-grade glioma (n=12), low-grade glioma (n=7), medulloblastoma (n=4), ATRT (n=2), and choroid plexus papilloma (n=1). Known cancer predisposition syndromes were identified or confirmed in 18 patients. Of these, the most common alterations were in TP53 (n=6), CHEK2 (2), NF1 (n=2), SMARCB1 (n=2), and PTEN (n=2). Patients were referred to genetic counseling in 26 cases and malignancy screenings were implemented in 25 cases. Germline findings affected malignancy treatment in 10 cases, most often through use of targeted therapeutics or avoidance of radiation. CONCLUSIONS: In our series, we found that 16% of pediatric neuro-oncology patients harbored germline mutations, the majority of which were associated with cancer predisposition syndromes. These results support standardizing screening for pathogenic germline mutations in pediatric neuro-oncology patients.

The evolving role of next generation sequencing in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal haematological disorders characterized by haematopoietic cell dysplasia, peripheral blood cytopenias, and a predisposition for developing acute myeloid leukaemia (AML). Cytogenetics have historically been important in diagnosis and prognosis in MDS, but the growing accessibility of next generation sequencing (NGS) has led to growing research in the roles of molecular genetic variation on clinical decision-making in these disorders. Multiple genes have been previously studied and found to be associated with specific outcomes or disease types within MDS and knowledge of mutations in these genes provides insight into previously defined MDS subtypes. Knowledge of these mutations also informs development of novel therapies in the treatment of MDS. The precise role of NGS in the diagnosis, prognosis and monitoring of MDS remains unclear but the improvements in NGS technology and accessibility affords clinicians an additional practice tool to provide the best care for patients.

ArcasHLA: high-resolution HLA typing from RNAseq.

MotivationThe human leukocyte antigen (HLA) locus plays a critical role in tissue compatibility and regulates the host response to many diseases, including cancers and autoimmune di3orders. Recent improvements in the quality and accessibility of next-generation sequencing have made HLA typing from standard short-read data practical. However, this task remains challenging given the high level of polymorphism and homology between HLA genes. HLA typing from RNA sequencing is further complicated by post-transcriptional modifications and bias due to amplification.ResultsHere, we present arcasHLA: a fast and accurate in silico tool that infers HLA genotypes from RNA-sequencing data. Our tool outperforms established tools on the gold-standard benchmark dataset for HLA typing in terms of both accuracy and speed, with an accuracy rate of 100% at two-field resolution for Class I genes, and over 99.7% for Class II. Furthermore, we evaluate the performance of our tool on a new biological dataset of 447 single-end total RNA samples from nasopharyngeal swabs, and establish the applicability of arcasHLA in metatranscriptome studies.Availability and implementationarcasHLA is available at https://github.com/RabadanLab/arcasHLA.Supplementary information Supplementary data are available at Bioinformatics online.

Use of Next Generation Sequencing Panel for Routine Diagnosis of Hereditary Hemolytic Anemias

Hereditary hemolytic anemia (HHA) are a heterogeneous group of disorders due to germline mutations of the red cell cytoskeleton (e.g. hereditary spherocytosis (HS) and hereditary elliptocytosis/pyropoikilocytosis (HE/HPP)) or enzyme deficiencies (e.g. glucose 6 phosphate dehydrogenase deficiency (G6PD) and pyruvate kinase deficiency (PKD). Routine morphological and biochemical analysis may be inconclusive in neonates due to the physiological nature of erythroid cell maturation and can also be misleading in transfusion-dependent patients. Additionally, there has been increasing awareness of inherited red cell membrane disorders that are not easily identified by routine laboratory approaches. For example, clinically insignificant defects of RBC membrane genes (e.g. alpha LELY and alpha LEPRA in SPTA1), which can be present in the parents without significant hemolysis, may result in compound heterozygosity in the offspring, causing severe morbidity or even mortality due to significant hemolysis. Awareness of these low expression alleles is important for genetic counseling purposes. Molecular studies, although becoming more mainstream, have not been used extensively to diagnose these disorders. This is most likely due to the complex genetic nature of these disorders (e.g. large genes with multiple exons involved, and multi-gene disorders (i.e. hyperbilirubinemia due to HS as well as involvement of genes involved in bilirubin metabolism). The accessibility of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible.Our current diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for NGS are performed using a Sure Select kit (Agilent). Indexed samples are quantified using qPCR and then pooled prior to sequencing on the Illumina NextSeq or HiSeq instruments. Samples are sequenced using 150 bp paired-end sequencing. This panel includes genes responsible for RBC membrane defects, enzyme deficiencies, as well as bilirubin uridine diphosphate glucuronosyltransferase (UGT1A) genes that have a distinct role in hyperbilirubinemia.We now report the first 268 patients evaluated using our NGS panel between 2015-2018. These patients were evaluated using an Institutional Review Board Protocol (IRB - 00077285). The age of the patients ranged from newborn to 68 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified according to the American College of Medical Genetics (ACMG) guidelines.We identified pathogenic and likely pathogenic variants in 64/268 (24%) patients that were clearly responsible for the disease phenotype (e.g. moderate to severe hemolytic anemia). Approximately half of them were novel mutations. Moreover, 29/268 (11%) of patients were homozygous for a promoter polymorphism in the UGT1A1 gene A(TA)7TAA (UGT1A1*28), which may lead to reduced expression of the UGT1A1 gene and Gilbert's syndrome. Furthermore, 4/29 UGT1A1 polymorphism cases were associated with pathogenic spectrin mutations, likely increasing the severity of the clinical phenotype in these patients. Overall, the most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits, followed by PKLR and ANK1 (Table 1). Complex interactions between variants in the SPTA1 gene and the common alpha-LELY and alpha-LEPRA alleles were predicted to be associated with HPP and autosomal recessive HS in 12/64 patients. Furthermore, 23/268 (9%) patients had mutations that were predicted to cause moderate to severe anemia if inherited with another mutation, making them important for genetic counseling purposes (data not shown). Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes. Many variants of unknown significance were also identified that could potentially contribute to disease.To conclude, the use of NGS provides a cost-effective and comprehensive method to assist in the diagnosis of hemolytic anemias, especially in instances where complex gene-gene interactions are suspected. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cancer genomics: why rare is valuable.

Rare conditions are sometimes ignored in biomedical research because of difficulties in obtaining specimens and limited interest from fund raisers. However, the study of rare diseases such as unusual cancers has again and again led to breakthroughs in our understanding of more common diseases. It is therefore unsurprising that with the development and accessibility of next-generation sequencing, much has been learnt from studying cancers that are rare and in particular those with uniform biological and clinical behavior. Herein, we describe how shotgun sequencing of cancers such as granulosa cell tumor, endometrial stromal sarcoma, epithelioid hemangioendothelioma, ameloblastoma, small-cell carcinoma of the ovary, clear-cell carcinoma of the ovary, nonepithelial ovarian tumors, chondroblastoma, and giant cell tumor of the bone has led to rapidly translatable discoveries in diagnostics and tumor taxonomies, as well as providing insights into cancer biology.