Guidelines for diagnostic next-generation sequencing

We present, on behalf of EuroGentest and the European Society of Human Genetics, guidelines for the evaluation and validation of next-generation sequencing (NGS) applications for the diagnosis of genetic disorders. The work was performed by a group of laboratory geneticists and bioinformaticians, and discussed with clinical geneticists, industry and patients'' representatives, and other stakeholders in the field of human genetics. The statements that were written during the elaboration of the guidelines are presented here. The background document and full guidelines are available as supplementary material. They include many examples to assist the laboratories in the implementation of NGS and accreditation of this service. The work and ideas presented by others in guidelines that have emerged elsewhere in the course of the past few years were also considered and are acknowledged in the full text. Interestingly, a few new insights that have not been cited before have emerged during the preparation of the guidelines. The most important new feature is the presentation of a ‘rating system'' for NGS-based diagnostic tests. The guidelines and statements have been applauded by the genetic diagnostic community, and thus seem to be valuable for the harmonization and quality assurance of NGS diagnostics in Europe.Next-generation sequencing (NGS) allows for the fast generation of thousands to millions of base pairs of DNA sequence of an individual patient. The relatively fast emergence and the great success of these technologies in research herald a new era in genetic diagnostics. However, the new technologies bring challenges, both at the technical level and in terms of data management, as well as for the interpretation of the results and for counseling. We believe that all these aspects warrant consideration of what the precise role of NGS in diagnostics will be, today and tomorrow. Before even embarking on acquisition of machines and skills for performing NGS in diagnostics, many issues have to be dealt with. It is in this context that we propose the guidelines. These guidelines mostly deal with NGS testing in the context of rare and mostly monogenic diseases. They mainly focus on the targeted analysis of gene panels, either through specific capture assays, or by extracting data from whole-exome sequencing. In principle, whole-genome sequencing may – and shortly will – also be used to extract similar information. In that case, the guidelines would still apply, but because whole-genome sequencing would also allow detecting other molecular features of disease, they would have to be extended accordingly.The different aspects of NGS and diagnostics were discussed during three workshops. The first took place in Leuven, 25–26 February 2013. The preliminary views were presented during the EuroGentest Scientific Meeting in Prague, 7–8 March 2013. The second was an editorial workshop in Leuven, 1–2 October 2013, where the different people involved in writing the document came together to discuss the layout of the document and prepare the first draft. The first draft was finalized prior to the third meeting in Nijmegen, 21–22 November, 2013. To the latter meeting, a larger group of stakeholders was invited. They were invited to comment on the draft, and on the statements presented therein. The comments were included in a new version, which was circulated among the editorial group, prior to publication on the EuroGentest and European Society of Human Genetics websites. Eventually, the document was presented to the Board of the European Society of Human Genetics, for endorsement. Endorsement was formally obtained on 1 July 2015.The statements that emerged during the writing of the guidelines are briefly presented in this paper. They are more extensively explained in the full version of the guidelines, available as supplementary material. The supplementary material also includes definitions, general recommendations and importantly, a number of practical examples and templates.     

ACMG clinical laboratory standards for next-generation sequencing

Next-generation sequencing technologies have been and continue to be deployed in clinical laboratories, enabling rapid transformations in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude, and continuous advances are being made. It is now feasible to analyze an individual’s near-complete exome or genome to assist in the diagnosis of a wide array of clinical scenarios. Next-generation sequencing technologies are also facilitating further advances in therapeutic decision making and disease prediction for at-risk patients. However, with rapid advances come additional challenges involving the clinical validation and use of these constantly evolving technologies and platforms in clinical laboratories. To assist clinical laboratories with the validation of next-generation sequencing methods and platforms, the ongoing monitoring of next-generation sequencing testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics has developed the following professional standards and guidelines.Genet Med15 9, 733–747.     

Solving the molecular diagnostic testing conundrum for Mendelian disorders in the era of next-generation sequencing: single-gene,gene panel,or exome/genome sequencing

Next-generation sequencing is changing the paradigm of clinical genetic testing. Today there are numerous molecular tests available, including single-gene tests, gene panels, and exome sequencing or genome sequencing. As a result, ordering physicians face the conundrum of selecting the best diagnostic tool for their patients with genetic conditions. Single-gene testing is often most appropriate for conditions with distinctive clinical features and minimal locus heterogeneity. Next-generation sequencing–based gene panel testing, which can be complemented with array comparative genomic hybridization and other ancillary methods, provides a comprehensive and feasible approach for heterogeneous disorders. Exome sequencing and genome sequencing have the advantage of being unbiased regarding what set of genes is analyzed, enabling parallel interrogation of most of the genes in the human genome. However, current limitations of next-generation sequencing technology and our variant interpretation capabilities caution us against offering exome sequencing or genome sequencing as either stand-alone or first-choice diagnostic approaches. A growing interest in personalized medicine calls for the application of genome sequencing in clinical diagnostics, but major challenges must be addressed before its full potential can be realized. Here, we propose a testing algorithm to help clinicians opt for the most appropriate molecular diagnostic tool for each scenario.Genet Med17 6, 444–451.     

Identification of false-negative mutations missed by next-generation sequencing in retinitis pigmentosa patients: a complementary approach to clinical genetic diagnostic testing

PurposeRetinitis pigmentosa (RP) is a major cause of heritable human blindness with extreme genetic heterogeneity. A large number of causative genes have been defined by next-generation sequencing (NGS). However, due to technical limitations, determining the existence of uncovered or low-depth regions is a fundamental challenge in analyzing NGS data. Therefore, undetected mutations may exist in genomic regions less effectively covered by NGS.MethodsTo address this problem, we tested a complementary approach for identifying previously undetected mutations in NGS data sets. The strategy consisted of coverage-based analysis and additional target screening of low-depth regions. Fifty RP patients were analyzed, and none of the mutations found had previously been identified by NGS.ResultsCoverage-based analysis indicated that, because of a highly repetitive sequence, the RPGR open reading frame (ORF)15 was located in an uncovered or low-depth region. Through additional screening of ORF15, we identified pathogenic mutations in 14% (7/50) of patients, including four novel mutations first described herein.ConclusionIn brief, we support the need for a complementary approach to identify mutations undetected by NGS, underscoring the power and significance of combining coverage-based analysis with additional target screening of low-depth regions in improving diagnosis of genetic diseases. In addition to its usefulness in RP, this approach is likely applicable to other Mendelian diseases.Genet Med 17 4, 307–311.     

Multiplex molecular testing for management of infectious gastroenteritis in a hospital setting: a comparative diagnostic and clinical utility study

Laboratory diagnosis and clinical management of inpatients with diarrhoea is complex and time consuming. Tests are often requested sequentially and undertaken in different laboratories. This causes prolonged unnecessary presumptive isolation of patients, because most cases are non-infectious. A molecular multiplex test (Luminex^® Gastrointestinal Pathogen Panel (GPP)) was compared with conventional testing over 8 months to determine diagnostic accuracy, turnaround times, laboratory costs, use of isolation facilities and user acceptability. A total of 262 (12%) patients had a pathogen detected by conventional methods compared with 483 (22.1%) by GPP. Most additional cases were detected in patients developing symptoms in the first 4 days of admission. Additional cases were detected because of presumed improved diagnostic sensitivity but also because clinicians had not requested the correct pathogen. Turnaround time (41.8 h) was faster than bacterial culture (66.5 h) and parasite investigation (66.5 h) but slower than conventional testing for Clostridium difficile (17.3 h) and viruses (27 h). The test could allow simplified requesting by clinicians and a consolidated laboratory workflow, reducing the overall number of specimens received by the laboratory. A total of 154 isolation days were saved at an estimated cost of £30 800. Consumables and labour were estimated at £150 641 compared with £63 431 for conventional testing. Multiplex molecular testing using a panel of targets allowed enhanced detection and a consolidated laboratory workflow. This is likely to be of greater benefit to cases that present within the first 4 days of hospital admission.     

EagleView: a genome assembly viewer for next-generation sequencing technologies

The emergence of high-throughput next-generation sequencing technologies (e.g., 454 Life Sciences [Roche], Illumina sequencing [formerly Solexa sequencing]) has dramatically sped up whole-genome de novo sequencing and resequencing. While the low cost of these sequencing technologies provides an unparalleled opportunity for genome-wide polymorphism discovery, the analysis of the new data types and huge data volume poses formidable informatics challenges for base calling, read alignment and genome assembly, polymorphism detection, as well as data visualization. We introduce a new data integration and visualization tool EagleView to facilitate data analyses, visual validation, and hypothesis generation. EagleView can handle a large genome assembly of millions of reads. It supports a compact assembly view, multiple navigation modes, and a pinpoint view of technology-specific trace information. Moreover, EagleView supports viewing coassembly of mixed-type reads from different technologies and supports integrating genome feature annotations into genome assemblies. EagleView has been used in our own lab and by over 100 research labs worldwide for next-generation sequence analyses. The EagleView software is freely available for not-for-profit use at http://bioinformatics.bc.edu/marthlab/EagleView.     

Using next-generation sequencing as a genetic diagnostic tool in rare autosomal recessive neurologic Mendelian disorders

Next-generation sequencing was used to investigate 9 rare Chinese pedigrees with rare autosomal recessive neurologic Mendelian disorders. Five probands with ataxia-telangectasia and 1 proband with chorea-acanthocytosis were analyzed by targeted gene sequencing. Whole-exome sequencing was used to investigate 3 affected individuals with Joubert syndrome, nemaline myopathy, or spastic ataxia Charlevoix-Saguenay type. A list of known and novel candidate variants was identified for each causative gene. All variants were genetically verified by Sanger sequencing or quantitative polymerase chain reaction with the strategy of disease segregation in related pedigrees and healthy controls. The advantages of using next-generation sequencing to diagnose rare autosomal recessive neurologic Mendelian disorders characterized by genetic and phenotypic heterogeneity are demonstrated. A genetic diagnostic strategy combining the use of targeted gene sequencing and whole-exome sequencing with the aid of next-generation sequencing platforms has shown great promise for improving the diagnosis of neurologic Mendelian disorders.     

Comparative of clinical performance between next-generation sequencing and standard blood culture diagnostic method in patients suffering from sepsis

BackgroundNext-generation sequencing (NGS) is a massively unbiased sequencing technology. The objective of this study was to evaluate the performance of NGS-based approach in the detection of microorganisms from septic patients and compare with results of blood culture (BC).MethodsThe observational and non-interventional study was conducted from April 2019 to August 2019.ResultsA total of 96 sets of BC and 48 NGS results obtained from 48 septic patients were analyzed in this study. Thirty-two microorganisms (27 bacteria, 3 fungi and 2 viral) were detected by NGS in 23 (47.9%) patients; and 18 bacteria in 18 (37.5%) patients by BC. Exclusion of skin commensals, the positivity of NGS and BC was 62.5% and 14.5%, respectively (P < 0.001). Microorganisms identified by NGS demonstrated positive agreement with BC in 12 (25%) patients, including concordant results in 11 (22.9%) cases, and discrepancy results in 1 (2%). Of 11 patients with concordant results, 4 had additional microorganisms detected by NGS. NGS-positive but BC-negative was found in 9 (18.7%) patients. Using NGS, difficult-to-culture micro-organisms such as Pneumocystic jirovecii was identified in 2 patients, and Leptospira interrogans in one. Six (12.5%) patients with BC-positive but NGS-negative, whereas skin commensals were isolated in 4 (66.6%) cases. The number of patients that were positive by BC only increase from 29% to 47.9% when combining NGS and BC analyses (P = 0.033).ConclusionsOur study support the advantage of NGS for the diagnosis of infecting microorganisms in sepsis, especially for microorganisms that are currently difficult or impossible to culture.     

Detection of structural variation using target captured next-generation sequencing data for genetic diagnostic testing

PurposeStructural variation (SV) is associated with inherited diseases. Next-generation sequencing (NGS) is an efficient method for SV detection because of its high-throughput, low cost, and base-pair resolution. However, due to lack of standard NGS protocols and a limited number of clinical samples with pathogenic SVs, comprehensive standards for SV detection, interpretation, and reporting are to be established.MethodsWe performed SV assessment on 60,000 clinical samples tested with hereditary cancer NGS panels spanning 48 genes. To evaluate NGS results, NGS and orthogonal methods were used separately in a blinded fashion for SV detection in all samples.ResultsA total of 1,037 SVs in coding sequence (CDS) or untranslated regions (UTRs) and 30,847 SVs in introns were detected and validated. Across all variant types, NGS shows 100% sensitivity and 99.9% specificity. Overall, 64% of CDS/UTR SVs were classified as pathogenic/likely pathogenic, and five deletions/duplications were reclassified as pathogenic using breakpoint information from NGS.ConclusionThe SVs presented here can be used as a valuable resource for clinical research and diagnostics. The data illustrate NGS as a powerful tool for SV detection. Application of NGS and confirmation technologies in genetic testing ensures delivering accurate and reliable results for diagnosis and patient care.     

Targeted next-generation sequencing of deafness genes in hearing-impaired individuals uncovers informative mutations

PurposeTargeted next-generation sequencing provides a remarkable opportunity to identify variants in known disease genes, particularly in extremely heterogeneous disorders such as nonsyndromic hearing loss. The present study attempts to shed light on the complexity of hearing impairment.MethodsUsing one of two next-generation sequencing panels containing either 80 or 129 deafness genes, we screened 30 individuals with nonsyndromic hearing loss (from 23 unrelated families) and analyzed 9 normal-hearing controls.ResultsOverall, we found an average of 3.7 variants (in 80 genes) with deleterious prediction outcome, including a number of novel variants, in individuals with nonsyndromic hearing loss and 1.4 in controls. By next-generation sequencing alone, 12 of 23 (52%) probands were diagnosed with monogenic forms of nonsyndromic hearing loss; one individual displayed a DNA sequence mutation together with a microdeletion. Two (9%) probands have Usher syndrome. In the undiagnosed individuals (10/23; 43%) we detected a significant enrichment of potentially pathogenic variants as compared to controls.ConclusionNext-generation sequencing combined with microarrays provides the diagnosis for approximately half of the GJB2 mutation–negative individuals. Usher syndrome was found to be more frequent in the study cohort than anticipated. The conditions in a proportion of individuals with nonsyndromic hearing loss, particularly in the undiagnosed group, may have been caused or modified by an accumulation of unfavorable variants across multiple genes.Genet Med16 12, 945–953.     

Full-length next-generation sequencing of HLA class I and II genes in a cohort from Thailand

The human leukocyte antigen (HLA) genes are highly variable and are known to play an important role in disease outcomes, including infectious diseases. Prior knowledge of HLA polymorphisms in a population usually forms the basis for an effective case-control study design. As a prelude to future disease association analyses, we report HLA class I and II diversity in 334 unrelated donors from a Dengue vaccine efficacy trial conducted in Thailand. Long-range PCR amplification of six HLA loci was performed on DNA extracted from saliva samples. HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1 were genotyped using a next-generation sequencing method presented at the 17th International HLA and Immunogenetics Workshop. In total, we identified 201 HLA alleles, including 35 HLA-A, 57 HLA-B, 28 HLA-C, 24 HLA-DPB1, 21 HLA-DQB1 and 36 HLA-DRB1 alleles. Very common HLA alleles with frequencies greater than 10 percent were A111:01:01, A133:03:01, A124:02:01, B146:01:01, C107:02:01, C101:02:01, C108:01:01, DPB1105:01:01, DPB1113:01:01, DPB1104:01:01, DPB1102:01:02, DQB1103:01:01, DQB1105:02:01, DQB1103:03:02, DRB1112:02:01, DRB1109:01:02, and DRB1115:02:01. A novel HLA allele, B115:450, had a non-synonymous substitution and occurred in more than one donor. Population-based full-length NGS HLA typing is more conclusive and provides a sound foundation for exploring disease association in a given population.     

The use of next-generation sequencing in clinical diagnosis of familial hypercholesterolemia

PurposeFamilial hypercholesterolemia is a common Mendelian disorder associated with early-onset coronary heart disease that can be treated by cholesterol-lowering drugs. The majority of cases in the United Kingdom are currently without a molecular diagnosis, which is partly due to the cost and time associated with standard screening techniques. The main purpose of this study was to test the sensitivity and specificity of two next-generation sequencing protocols for genetic diagnosis of familial hypercholesterolemia.MethodsLibraries were prepared for next-generation sequencing by two target enrichment protocols; one using the SureSelect Target Enrichment System and the other using the PCR-based Access Array platform.ResultsIn the validation cohort, both protocols showed 100% specificity, whereas the sensitivity for short variant detection was 100% for the SureSelect Target Enrichment and 98% for the Access Array protocol. Large deletions/duplications were only detected using the SureSelect Target Enrichment protocol. In the prospective cohort, the mutation detection rate using the Access Array was highest in patients with clinically definite familial hypercholesterolemia (67%), followed by patients with possible familial hypercholesterolemia (26%).ConclusionWe have shown the potential of target enrichment methods combined with next-generation sequencing for molecular diagnosis of familial hypercholesterolemia. Adopting these assays for patients with suspected familial hypercholesterolemia could improve cost-effectiveness and increase the overall number of patients with a molecular diagnosis.Genet Med15 12, 948–957.     

Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas.     

Targeted next-generation sequencing as a comprehensive test for patients with and female carriers of DMD/BMD: a multi-population diagnostic study

Duchenne and Becker muscular dystrophies (DMD/BMD) are the most commonly inherited neuromuscular disease. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of the dystrophin gene and complex causative mutation spectrum. Such traditional methods as multiplex ligation-dependent probe amplification plus Sanger sequencing require multiple steps to fulfill the diagnosis of DMD/BMD. Here, we introduce a new single-step method for the genetic analysis of DMD patients and female carriers in real clinical settings and demonstrate the validation of its accuracy. A total of 89 patients, 18 female carriers and 245 non-DMD patients were evaluated using our targeted NGS approaches. Compared with traditional methods, our new method yielded 99.99% specificity and 98.96% sensitivity for copy number variations detection and 100% accuracy for the identification of single-nucleotide variation mutations. Additionally, this method is able to detect partial deletions/duplications, thus offering precise personal DMD gene information for gene therapy. We detected novel partial deletions of exons in nine samples for which the breakpoints were located within exonic regions. The results proved that our new method is suitable for routine clinical practice, with shorter turnaround time, higher accuracy, and better insight into comprehensive genetic information (detailed breakpoints) for ensuing gene therapy.     

Development of a consent resource for genomic data sharing in the clinical setting

PurposeData sharing between clinicians, laboratories, and patients is essential for improvements in genomic medicine, but obtaining consent for individual-level data sharing is often hindered by a lack of time and resources. To address this issue, the Clinical Genome Resource (ClinGen) developed tools to facilitate consent, including a one-page consent form and online supplemental video with information on key topics, such as risks and benefits of data sharing.MethodsTo determine whether the consent form and video accurately conveyed key data sharing concepts, we surveyed 5,162 members of the general public. We measured comprehension at baseline, after reading the form and watching the video. Additionally, we assessed participants’ attitudes toward genomic data sharing.ResultsParticipants’ performance on comprehension questions significantly improved over baseline after reading the form and continued to improve after watching the video.ConclusionResults suggest reading the form alone provided participants with important knowledge regarding broad data sharing, and watching the video allowed for broader comprehension. These materials are now available at http://www.clinicalgenome.org/share. These resources will provide patients a straightforward way to share their genetic and health information, and improve the scientific community’s access to data generated through routine healthcare.