Advances in DNA sequencing technologies for high resolution HLA typing

This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms – ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing.     

Challenges in the application of NGS in the clinical laboratory

Next-generation sequencing (NGS), also known as massively parallel sequencing, has revolutionized genomic research. The current advances in NGS technology make it possible to provide high resolution, high throughput HLA typing in clinical laboratories. The focus of this review is on the recent development and implementation of NGS in clinical laboratories. Here, we examine the critical role of NGS technologies in clinical immunology for HLA genotyping. Two major NGS platforms (Illumina and Ion Torrent) are characterized including NGS library preparation, data analysis, and validation. Challenges of NGS implementation in the clinical laboratory are also discussed, including sequencing error rate, bioinformatics, result interpretation, analytic sensitivity, as well as large data storage. This review aims to promote the broader applications of NGS technology in clinical laboratories and advocate for the novel applications of NGS to drive future research.     

Next-generation sequencing for constitutional variants in the clinical laboratory, 2021 revision: a technical standard of the American College of Medical Genetics and Genomics (ACMG)

Next-generation sequencing (NGS) technologies are now established in clinical laboratories as a primary testing modality in genomic medicine. These technologies have reduced the cost of large-scale sequencing by several orders of magnitude. It is now cost-effective to analyze an individual with disease-targeted gene panels, exome sequencing, or genome sequencing to assist in the diagnosis of a wide array of clinical scenarios. While clinical validation and use of NGS in many settings is established, there are continuing challenges as technologies and the associated informatics evolve. To assist clinical laboratories with the validation of NGS methods and platforms, the ongoing monitoring of NGS testing to ensure quality results, and the interpretation and reporting of variants found using these technologies, the American College of Medical Genetics and Genomics (ACMG) has developed the following technical standards.     

Next-generation sequencing technology in clinical virology

Recent advances in nucleic acid sequencing technologies, referred to as ‘next-generation’ sequencing (NGS), have produced a true revolution and opened new perspectives for research and diagnostic applications, owing to the high speed and throughput of data generation. So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics. These applications are particularly suitable for viruses such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, whose error-prone replication machinery, combined with the high replication rate, results, in each infected individual, in the formation of many genetically related viral variants referred to as quasi-species. The viral quasi-species, in turn, represents the substrate for the selective pressure exerted by the immune system or by antiviral drugs. With traditional approaches, it is difficult to detect and quantify minority genomes present in viral quasi-species that, in fact, may have biological and clinical relevance. NGS provides, for each patient, a dataset of clonal sequences that is some order of magnitude higher than those obtained with conventional approaches. Hence, NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harboured by different cell lineages, the dynamics of development of drug resistance, and the re-emergence of hidden genomes after treatment interruptions. The diagnostic application of NGS is just around the corner.     

Identification and characterization of novel HLA alleles: Utility of next-generation sequencing methods

The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology.     

Next generation sequencing to determine HLA class II genotypes in a cohort of hematopoietic cell transplant patients and donors

Current high-resolution HLA typing technologies frequently produce ambiguous results that mandate extended testing prior to reporting. Through multiplex sequencing of individual amplicons from many individuals at multiple loci, next generation sequencing (NGS) promises to eliminate heterozygote ambiguities and extend the breadth of genetic data acquired with little additional effort. We report here on assessment of a novel NGS HLA genotyping system for resequencing exons 2 and 3 of DRB1/B3/B4/B5, DQA1 and DQB1 and exon 2 of DPA1 and DPB1 on the MiSeq platform. In a cohort of 2605 hematopoietic cell transplant recipients and donors, NGS achieved 99.6% accuracy for DRB1 allele assignments and 99.5% for DQB1, compared to legacy genotypes generated pretransplant. NGS provided at least single 4-digit allele resolution for 97% of genotypes at DRB1 and 100% at DQB1. Overall, NGS typing identified 166 class II alleles, including 9 novel sequences with greater than 99% accuracy for DRB1 and DQB1 genotypes and elimination of diploid ambiguities through in-phase sequencing demonstrated the robust reliability of the NGS HLA genotyping reagents and analysis software employed in this study.     

Studying the epigenome using next generation sequencing

The advances in next generation sequencing (NGS) technologies have had a significant impact on epigenomic research. The arrival of NGS technologies has enabled a more powerful sequencing based method--that is, ChIP-Seq--to interrogate whole genome histone modifications, improving on the conventional microarray based method (ChIP-chip). Similarly, the first human DNA methylome was mapped using NGS technologies. More importantly, studies of DNA methylation and histone modification using NGS technologies have yielded new discoveries and improved our knowledge of human biology and diseases. The concept that cytosine methylation was restricted to CpG dinucleotides has only been recently challenged by new data generated from sequencing the DNA methylome. Approximately 25% of all cytosine methylation identified in stem cells was in a non-CG context. The non-CG methylation was more enriched in gene bodies and depleted in protein binding sites and enhancers. The recent developments of third generation sequencing technologies have shown promising results of directly sequencing methylated nucleotides and having the ability to differentiate between 5-methylcytosine and 5-hydroxymethylcytosine. The importance of 5-hydroxymethylcytosine remains largely unknown, but it has been found in various tissues. 5-hydroxymethylcytosine was particularly enriched at promoters and in intragenic regions (gene bodies) but was largely absent from non-gene regions in DNA from human brain frontal lobe tissue. The presence of 5-hydroxymethylcytosine in gene bodies was more positively correlated with gene expression levels. The importance of studying 5-methylcytosine and 5-hydroxymethylcytosine separately for their biological roles will become clearer when more efficient methods to distinguish them are available.     

Lake Louise Mutation Detection Meeting 2013: Clinical Translation of Next‐Generation Sequencing Requires Optimization of Workflows and Interpretation of Variants

With the exponential reduction of the cost of next‐generation sequencing (NGS), it is no longer the generation of data but the analysis and interpretation of massive amounts of sequencing data that are seen as key challenges for the effective integration of these technologies into clinical practice. Clinical geneticists, informaticians, and scientists from 17 countries gathered for the 12th International Symposium on Mutation in the Genome at the Fairmont Chateau Lake Louise (Canada) to discuss technological advances and applications of NGS and consider possible approaches to the challenges of clinical translation. Here, we provide an overview of the main themes of the meeting that included development of innovative solutions for variant sharing, tools and resources for NGS analysis, novel technology and methodology development, NGS‐based discovery of disease pathogenesis, development of multigene NGS sequencing panels for clinical use, exploring diagnostic utility of whole‐exome and whole‐genome sequencing, and, finally, integration of genomic sequencing into the clinic.     

Efficient ten-gene analysis of NSCLC tissue samples by next-generation sequencing

In the era of personalized medicine, lung cancer is a typical disease which can be treated strategically based on the patient’s histological and molecular diagnosis. Immunohistochemistry (IHC), fluorescence in-situ hybridization (FISH), Sanger sequencing and real-time PCR are techniques commonly used in clinical laboratories. Many patients are required to use several of the above technologies to get a complete diagnosis, which is expensive and timeconsuming. Next generation of sequencing (NGS) has the advantage to simultaneously analyze multigene mutations. The average cost for each patient is affordable if each run contains a certain number of samples. In this study, we tested a 10-gene, 32-mutation detection NGS method, which was used to test 195 samples from non-small cell lung cancer (NSCLC). Sanger sequencing and Amplification-refractory Mutation System (AMRS) PCR were employed to verify Epidermal Growth Factor Receptor (EGFR) and Anaplastic Lymphoma Kinase (ALK) results. This NGS method was partially proved to have a higher sensitivity to detect mutations with low abundance than Sanger sequencing and even ARMS PCR. Using genomic DNA to detect gene fusions may have some disadvantages to miss low abundance or large fragment fusions. As compared to using a few different technologies to analyze multigene mutations, small NGS analysis panel is a clinically applicable, efficient and affordable choice for NSCLC patients.     

Isolation and next generation sequencing of archival formalin-fixed DNA

DNA from archived organs is presumed unsuitable for genomic studies because of excessive formalin-fixation. As next generation sequencing (NGS) requires short DNA fragments, and Uracil-N-glycosylase (UNG) can be used to overcome deamination, there has been renewed interest in the possibility of genomic studies using these collections. We describe a novel method of DNA extraction capable of providing PCR amplicons of at least 400 bp length from such excessively formalin-fixed human tissues. When compared with a leading commercial formalin-fixed DNA extraction kit, our method produced greater yields of DNA and reduced sequence variations. Analysis of PCR products using bacterial sub-cloning and Sanger sequencing from UNG-treated DNA unexpectedly revealed increased sequence variations, compared with untreated samples. Finally, whole exome NGS was performed on a myocardial sample fixed in formalin for 2 years and compared with lymphocyte-derived DNA (as a gold standard) from the same patient. Despite the reduction in the number and quality of reads in the formalin-fixed DNA, we were able to show that bioinformatic processing by joint calling and variant quality score recalibration (VQSR) increased the sensitivity four-fold to 56% and doubled specificity to 68% when compared with a standard hard-filtering approach. Thus, high-quality DNA can be extracted from excessively formalin-fixed tissues and bioinformatic processing can optimise sensitivity and specificity of results. Sequencing of several sub-cloned amplicons is an important methodological step in assessing DNA quality.     

Next-generation sequencing approaches and challenges in the diagnosis of developmental anomalies and intellectual disability

Recent advances in next-generation sequencing (NGS) technologies have revolutionized the field of human genetics. Alongside a broad panel of bioinformatics tools and databases, NGS technologies have unprecedentedly improved the molecular diagnosis rate and the identification of new genes associated with rare disorders. However, about 50% of patients remain without a final diagnosis. Here, we highlight the utility of NGS applications in developmental anomalies and intellectual disability, illustrating their main advantages and pitfalls. Through specific examples, we suggest novel strategies and tools for identifying the molecular bases in the remaining patients, and we outline future challenges.     

Collection and storage of HLA NGS genotyping data for the 17th International HLA and Immunogenetics Workshop

For over 50?years, the International HLA and Immunogenetics Workshops (IHIW) have advanced the fields of histocompatibility and immunogenetics (H&I) via community sharing of technology, experience and reagents, and the establishment of ongoing collaborative projects. Held in the fall of 2017, the 17th IHIW focused on the application of next generation sequencing (NGS) technologies for clinical and research goals in the H&I fields. NGS technologies have the potential to allow dramatic insights and advances in these fields, but the scope and sheer quantity of data associated with NGS raise challenges for their analysis, collection, exchange and storage. The 17th IHIW adopted a centralized approach to these issues, and we developed the tools, services and systems to create an effective system for capturing and managing these NGS data. We worked with NGS platform and software developers to define a set of distinct but equivalent NGS typing reports that record NGS data in a uniform fashion. The 17th IHIW database applied our standards, tools and services to collect, validate and store those structured, multi-platform data in an automated fashion. We have created community resources to enable exploration of the vast store of curated sequence and allele-name data in the IPD-IMGT/HLA Database, with the goal of creating a long-term community resource that integrates these curated data with new NGS sequence and polymorphism data, for advanced analyses and applications.     

Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory

The identification and/or prediction of the antimicrobial resistance of microorganisms in clinical materials solely by molecular means in the diagnostic microbiology laboratory is not novel. However, the ability to sequence multitudes of bacterial genomes and deliver and interpret the resultant sequence information in near "real-time" is the basis of next-generation sequencing (NGS) technologies. There have been numerous applications and successes of NGS applications in the clinical and public health domain. However, none have, as yet, delivered perhaps the most sought after application, i.e., the generation of microbial sequence data for "real-time" patient management. In this review, we discuss the use of NGS and whole-genome sequencing (WGS) of microorganisms as a logical next step for the routine diagnosis of infection and the prediction of antimicrobial susceptibility in the clinical microbiology laboratory.     

NGS and blood group systems: State of the art and perspectives

Molecular analysis, or genotyping, of genes involved in the expression of blood group antigens has been a standard strategy used in immunohaematology laboratories routinely. For the past ten years, next-generation sequencing (NGS), or second-generation sequencing, has become the reference method in genetics. Extensive study of distinct targets, large genomic regions, and even whole genome is henceforth possible by this approach at minimal cost. Blood group genotyping has thus taken advantage of this technological advent. A few preliminary studies have open the way to NGS in this field by studying one or several genes, in a wide range of samples (donors and patients) by using several different platforms. These works have helped in the identification of both the benefits and limitations of the technology. Other recently published studies have benefited from these preliminary data to improve the methodology, specificity and accuracy of output data. In parallel novel strategies, i.e. third-generation sequencing, which can sequence long DNA regions at the single-molecule level, have emerged and shown promise for the potential resolution of complex rearrangements involving genes of the Rh and MNS blood group systems respectively. As technological and methodological hurdles have been overcome, these approaches may be used in a clinical situation in a near future.     

New challenges,new opportunities: Next generation sequencing and its place in the advancement of HLA typing

The Human Leukocyte Antigen (HLA) system has a critical role in immunorecognition, transplantation, and disease association. Early typing techniques provided the foundation for genotyping methods that revealed HLA as one of the most complex, polymorphic regions of the human genome. Next Generation Sequencing (NGS), the latest molecular technology introduced in clinical tissue typing laboratories, has demonstrated advantages over other established methods. NGS offers high-resolution sequencing of entire genes in time frames and price points considered unthinkable just a few years ago, contributing a wealth of data informing histocompatibility assessment and standards of clinical care. Although the NGS platforms share a high-throughput massively parallel processing model, differing chemistries provide specific strengths and weaknesses. Research-oriented Third Generation Sequencing and related advances in bioengineering continue to broaden the future of NGS in clinical settings. These diverse applications have demanded equally innovative strategies for data management and computational bioinformatics to support and analyze the unprecedented volume and complexity of data generated by NGS. We discuss some of the challenges and opportunities associated with NGS technologies, providing a comprehensive picture of the historical developments that paved the way for the NGS revolution, its current state and future possibilities for HLA typing.     

De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer

Novel high-throughput DNA sequencing technologies allow researchers to characterize a bacterial genome during a single experiment and at a moderate cost. However, the increase in sequencing throughput that is allowed by using such platforms is obtained at the expense of individual sequence read length, which must be assembled into longer contigs to be exploitable. This study focuses on the Illumina sequencing platform that produces millions of very short sequences that are 35 bases in length. We propose a de novo assembler software that is dedicated to process such data. Based on a classical overlap graph representation and on the detection of potentially spurious reads, our software generates a set of accurate contigs of several kilobases that cover most of the bacterial genome. The assembly results were validated by comparing data sets that were obtained experimentally for Staphylococcus aureus strain MW2 and Helicobacter acinonychis strain Sheeba with that of their published genomes acquired by conventional sequencing of 1.5- to 3.0-kb fragments. We also provide indications that the broad coverage achieved by high-throughput sequencing might allow for the detection of clonal polymorphisms in the set of DNA molecules being sequenced.