Development and Validation of Clinical Whole-Exome and Whole-Genome Sequencing for Detection of Germline Variants in Inherited Disease

Context.-With the decrease in the cost of sequencing, the clinical testing paradigm has shifted from single gene to gene panel and now whole-exome and whole-genome sequencing. Clinical laboratories are rapidly implementing next-generation sequencing-based whole-exome and whole-genome sequencing. Because a large number of targets are covered by whole-exome and whole-genome sequencing, it is critical that a laboratory perform appropriate validation studies, develop a quality assurance and quality control program, and participate in proficiency testing. Objective.-To provide recommendations for wholeexome and whole-genome sequencing assay design, validation, and implementation for the detection of germline variants associated in inherited disorders. Data Sources.-An example of trio sequencing, filtration and annotation of variants, and phenotypic consideration to arrive at clinical diagnosis is discussed. Conclusions.-It is critical that clinical laboratories planning to implement whole-exome and whole-genome sequencing design and validate the assay to specifications and ensure adequate performance prior to implementation. Test design specifications, including variant filtering and annotation, phenotypic consideration, guidance on consenting options, and reporting of incidental findings, are provided. These are important steps a laboratory must take to validate and implement whole-exome and whole-genome sequencing in a clinical setting for germline variants in inherited disorders

Development and Validation of Targeted Next-Generation Sequencing Panels for Detection of Germline Variants in Inherited Diseases.

Context.-The number of targeted next-generation sequencing (NGS) panels for genetic diseases offered by clinical laboratories is rapidly increasing. Before an NGS-based test is implemented in a clinical laboratory, appropriate validation studies are needed to determine the performance characteristics of the test. Objective.-To provide examples of assay design and validation of targeted NGS gene panels for the detection of germline variants associated with inherited disorders. Data Sources.-The approaches used by 2 clinical laboratories for the development and validation of targeted NGS gene panels are described. Important design and validation considerations are examined. Conclusions.-Clinical laboratories must validate performance specifications of each test prior to implementation. Test design specifications and validation data are provided, outlining important steps in validation of targeted NGS panels by clinical diagnostic laboratories

Adenosine-5′-phosphosulfate - a multifaceted modulator of bifunctional 3′-phospho-adenosine-5′-phosphosulfate synthases and related enzymes

All sulfation reactions rely on active sulfate in the form of 3′-phosphoadenosine-5′-phosphosulfate (PAPS). In fungi, bacteria, and plants, the enzymes responsible for PAPS synthesis, ATP sulfurylase and adenosine-5′-phosphosulfate (APS) kinase, reside on separate polypeptide chains. In metazoans, however, bifunctional PAPS synthases catalyze the consecutive steps of sulfate activation by converting sulfate to PAPS via the intermediate APS. This intricate molecule and the related nucleotides PAPS and 3′-phospho-adenosine-5′-phosphate modulate the function of various enzymes from sulfation pathways, and these effects are summarized in this review. On the ATP sulfurylase domain that initially produces APS from sulfate and ATP, APS acts as a potent product inhibitor, being competitive with both ATP and sulfate. For the APS kinase domain that phosphorylates APS to PAPS, APS is an uncompetitive substrate inhibitor that can bind both at the ATP/ADP binding site and the PAPS/APS-binding site. For human PAPS synthase 1, the steady-state concentration of APS has been modelled to be 1.6 lM, but this may increase up to 60 lM under conditions of sulfate excess. It is noteworthy that the APS concentration for maximal APS kinase activity is 15 lM. Finally, we recognized APS as a highly specific stabilizer of bifunctional PAPS synthases. APS most likely stabilizes the APS kinase part of these proteins by forming a dead-end enzyme–ADP–APS complex at APS concentrations between 0.5 and 5 lM; at higher concentrations, APS may bind to the catalytic centers of ATP sulfurylase. Based on the assumption that cellular concentrations of APS fluctuate within this range, APS can therefore be regarded as a key modulator of PAPS synthase functions

Identification and Characterization of MicroRNAs in Normal Equine Tissues by Next Generation Sequencing

The role of microRNAs (miRNAs) as a post-transcriptional gene regulator has been elucidated in a broad range of organisms including domestic animals. Characterization of miRNAs in normal tissues is an important step to investigate the functions of miRNAs in various physiological and pathological conditions. Using Illumina Next Generation Sequencing (NGS) technology, we identified a total of 292 known and 329 novel miRNAs in normal horse tissues including skeletal muscle, colon and liver. Distinct sets of miRNAs were differentially expressed in a tissue-specific manner. The miRNA genes were distributed across all the chromosomes except chromosomes 29 and 31 in the horse reference genome. In some chromosomes, multiple miRNAs were clustered and considered to be polycistronic transcript. A base composition analysis showed that equine miRNAs had a higher frequency of A+U than G+C. Furthermore, U tended to be more frequent at the 59 end of miRNA sequences. This is the first experimental study that identifies and characterizes the global miRNA expression profile in normal horse tissues. The present study enriches the horse miRNA database and provides useful information for further research dissecting biological functions of miRNAs in horse.open2

Determination of RET Sequence Variation in an MEN2 Unaffected Cohort Using Multiple-Sample Pooling and Next-Generation Sequencing

Multisample, nonindexed pooling combined with next-generation sequencing (NGS) was used to discover RET proto-oncogene sequence variation within a cohort known to be unaffected by multiple endocrine neoplasia type 2 (MEN2). DNA samples (113 Caucasians, 23 persons of other ethnicities) were amplified for RET intron 9 to intron 16 and then divided into 5 pools of <30 samples each before library prep and NGS. Two controls were included in this study, a single sample and a pool of 50 samples that had been previously sequenced by the same NGS methods. All 59 variants previously detected in the 50-pool control were present. Of the 61 variants detected in the unaffected cohort, 20 variants were novel changes. Several variants were validated by high-resolution melting analysis and Sanger sequencing, and their allelic frequencies correlated well with those determined by NGS. The results from this unaffected cohort will be added to the RET MEN2 database

Comparison of alignment software for genome-wide bisulphite sequence data

Recent advances in next generation sequencing (NGS) technology now provide the opportunity to rapidly interrogate the methylation status of the genome. However, there are challenges in handling and interpretation of the methylation sequence data because of its large volume and the consequences of bisulphite modification. We sequenced reduced representation human genomes on the Illumina platform and efficiently mapped and visualized the data with different pipelines and software packages. We examined three pipelines for aligning bisulphite converted sequencing reads and compared their performance. We also comment on pre-processing and quality control of Illumina data. This comparison highlights differences in methods for NGS data processing and provides guidance to advance sequence-based methylation data analysis for molecular biologists

Ikaros family zinc finger 1 regulates dendritic cell development and function in humans

Ikaros family zinc finger 1 (IKZF1) is a haematopoietic transcription factor required for mammalian B-cell development. IKZF1 deficiency also reduces plasmacytoid dendritic cell (pDC) numbers in mice, but its effects on human DC development are unknown. Here we show that heterozygous mutation of IKZF1 in human decreases pDC numbers and expands conventional DC1 (cDC1). Lenalidomide, a drug that induces proteosomal degradation of IKZF1, also decreases pDC numbers in vivo, and reduces the ratio of pDC/cDC1 differentiated from progenitor cells in vitro in a dose-dependent manner. In addition, non-classical monocytes are reduced by IKZF1 deficiency in vivo. DC and monocytes from patients with IKZF1 deficiency or lenalidomide-treated cultures secrete less IFN-alpha, TNF and IL-12. These results indicate that human DC development and function are regulated by IKZF1, providing further insights into the consequences of IKZF1 mutation on immune function and the mechanism of immunomodulation by lenalidomide

College of American Pathologists\u27 Laboratory Standards for Next-Generation Sequencing Clinical Tests

Context.-The higher throughput and lower per-base cost of next-generation sequencing (NGS) as compared to Sanger sequencing has led to its rapid adoption in clinical testing. The number of laboratories offering NGS-based tests has also grown considerably in the past few years, despite the fact that specific Clinical Laboratory Improvement Amendments of 1988/College of American Pathologists (CAP) laboratory standards had not yet been developed to regulate this technology. Objective.-To develop a checklist for clinical testing using NGS technology that sets standards for the analytic wet bench process and for bioinformatics or \u27\u27 dry bench\u27\u27 analyses. As NGS-based clinical tests are new to diagnostic testing and are of much greater complexity than traditional Sanger sequencing-based tests, there is an urgent need to develop new regulatory standards for laboratories offering these tests. Design.-To develop the necessary regulatory framework for NGS and to facilitate appropriate adoption of this technology for clinical testing, CAP formed a committee in 2011, the NGS Work Group, to deliberate upon the contents to be included in the checklist. Results.-A total of 18 laboratory accreditation checklist requirements for the analytic wet bench process and bioinformatics analysis processes have been included within CAP\u27s molecular pathology checklist (MOL). Conclusions.-This report describes the important issues considered by the CAP committee during the development of the new checklist requirements, which address documentation, validation, quality assurance, confirmatory testing, exception logs, monitoring of upgrades, variant interpretation and reporting, incidental findings, data storage, version traceability, and data transfer confidentiality

Analysing 454 amplicon resequencing experiments using the modular and database oriented Variant Identification Pipeline

<p>Abstract</p> <p>Background</p> <p>Next-generation amplicon sequencing enables high-throughput genetic diagnostics, sequencing multiple genes in several patients together in one sequencing run. Currently, no open-source out-of-the-box software solution exists that reliably reports detected genetic variations and that can be used to improve future sequencing effectiveness by analyzing the PCR reactions.</p> <p>Results</p> <p>We developed an integrated database oriented software pipeline for analysis of 454/Roche GS-FLX amplicon resequencing experiments using Perl and a relational database. The pipeline enables variation detection, variation detection validation, and advanced data analysis, which provides information that can be used to optimize PCR efficiency using traditional means. The modular approach enables customization of the pipeline where needed and allows researchers to adopt their analysis pipeline to their experiments. Clear documentation and training data is available to test and validate the pipeline prior to using it on real sequencing data.</p> <p>Conclusions</p> <p>We designed an open-source database oriented pipeline that enables advanced analysis of 454/Roche GS-FLX amplicon resequencing experiments using SQL-statements. This modular database approach allows easy coupling with other pipeline modules such as variant interpretation or a LIMS system. There is also a set of standard reporting scripts available.</p

Mitochondrial DNA Variant Discovery and Evaluation in Human Cardiomyopathies through Next-Generation Sequencing

Mutations in mitochondrial DNA (mtDNA) may cause maternally-inherited cardiomyopathy and heart failure. In homoplasmy all mtDNA copies contain the mutation. In heteroplasmy there is a mixture of normal and mutant copies of mtDNA. The clinical phenotype of an affected individual depends on the type of genetic defect and the ratios of mutant and normal mtDNA in affected tissues. We aimed at determining the sensitivity of next-generation sequencing compared to Sanger sequencing for mutation detection in patients with mitochondrial cardiomyopathy. We studied 18 patients with mitochondrial cardiomyopathy and two with suspected mitochondrial disease. We “shotgun” sequenced PCR-amplified mtDNA and multiplexed using a single run on Roche's 454 Genome Sequencer. By mapping to the reference sequence, we obtained 1,300× average coverage per case and identified high-confidence variants. By comparing these to >400 mtDNA substitution variants detected by Sanger, we found 98% concordance in variant detection. Simulation studies showed that >95% of the homoplasmic variants were detected at a minimum sequence coverage of 20× while heteroplasmic variants required >200× coverage. Several Sanger “misses” were detected by 454 sequencing. These included the novel heteroplasmic 7501T>C in tRNA serine 1 in a patient with sudden cardiac death. These results support a potential role of next-generation sequencing in the discovery of novel mtDNA variants with heteroplasmy below the level reliably detected with Sanger sequencing. We hope that this will assist in the identification of mtDNA mutations and key genetic determinants for cardiomyopathy and mitochondrial disease