Application of Next-Generation Sequencing in the Era of Precision Medicine

Next-generation sequencing (NGS) technologies represented the next step in the evolution of DNA sequencing, through the generation of thousands to millions of DNA sequences in a short time. The relatively fast emergence and success of NGS in research revolutionized the field of genomics and medical diagnosis. The traditional medicine model of diagnosis has changed to one precision medicine model, leading to a more accurate diagnosis of human diseases and allowing the selection of molecular target drugs for individual treatment. This chapter attempts to review the main features of NGS technique (concepts, data analysis, applications, advances and challenges), starting with a brief history of DNA sequencing followed by a comprehensive description of most used NGS platforms. Further topics will highlight the application of NGS towards routine practice, including variant detection, whole-exome sequencing (WES), whole-genome sequencing (WGS), custom panels (multi-gene), RNA-seq and epigenetic. The potential use of NGS in precision medicine is vast and a better knowledge of this technique is necessary for an efficacious implementation in the clinical workplace. A centralized chapter describing the main NGS aspects in the clinic could help beginners, scientists, researchers and health care professionals, as they will be responsible for translating genomic data into genomic medicine

Avaliação e validação da utilidade clínica do sequenciamento de nova geração (NGS) para confirmação do diagnóstico de doenças lisossômicas selecionadas

Introdução: As doenças lisossômicas (DLs) são patologias genéticas que, apesar de serem classificadas como raras, acometem uma significativa porcentagem da população. Muitos fatores tornam seu diagnóstico desafiador, entre eles a variabilidade no fenótipo, com poucos sinais e sintomas clínicos específicos. O desenvolvimento de ferramentas inovadoras de investigação, destacando-se os novos métodos de sequenciamento massivo, reduziria a “odisseia diagnóstica” enfrentada pelo paciente e sua família, proporcionaria um diagnóstico mais precoce com melhor resultado no tratamento nas situações em que há terapia disponível, além de um adequado aconselhamento genético. A tecnologia de sequenciamento de nova geração (next-generation sequencing, NGS) tem claras vantagens sobre as técnicas de sequenciamento convencional, oferecendo um alto rendimento diagnóstico ao permitir definir um espectro mutacional abrangente. O NGS permite o sequenciamento de vários genes simultaneamente com custo global relativamente baixo, tornando painéis de genes uma alternativa atrativa para o screening genético. Esta abordagem é capaz de detectar, de maneira altamente específica, variantes missense, nonsense, de sítio de splicing, e pequenas indels, e algumas grandes deleções em homozigose ou hemizigose. No entanto, para que essa tecnologia seja aplicável à prática clínica, é necessária uma etapa de validação prévia para a determinação dos parâmetros críticos de análise desde o processamento da amostra até a análise e interpretação de dados. Após a validação dos métodos, será possível a avaliação da utilidade clínica de painéis NGS para diversas aplicações, incluindo seu uso como método confirmatório nos casos de triagem neonatal alterada para doenças lisossômicas. Objetivos: 1) Desenhar e validar uma estratégia baseada no NGS para a análise de 24 genes, incluídos em 3 painéis distintos de acordo com parâmetros pré definidos, e associados com 22 DLs; 2) Avaliar a qualidade e eficiência do ADN extraído de sangue impregnado em papel filtro (SIPF) para uso no NGS; 3) Avaliar a utilidade clínica do NGS para: a) diagnóstico molecular, b) diagnóstico diferencial e, c) confirmação diagnóstica de casos alterados na triagem neonatal para pelo menos 4 dessas condições (doença de Gaucher, Fabry, Pompe e Niemann-Pick tipo A/B). Metodologia: Estudo descritivo, amostragem por conveniência, incluindo pacientes com diagnóstico clínico e bioquímico das DLs estudadas. Foi utilizada a plataforma NGS Ion Torrent Personal Genome Machine (Thermo Fisher Scientific) para sequenciamento de três painéis de genes, desenhados através da ferramenta Ion AmpliseqTM Designer (Thermo Fisher Scientific). Para a validação foram incluídos pacientes com diagnóstico molecular prévio por sequenciamento de Sanger. Para a extração de ADN de SIPF, foi avaliado o método não comercial de fenol-clorofórmio. A utilidade clínica do NGS foi estabelecida através do: a) diagnóstico molecular de um grupo de pacientes com suspeita de lipofuscinose ceróide neuronal tipo 2 (CLN2) para o estabelecimento do genótipo (TPP1) associado à doença, assim como estabeleceu-se o diagnóstico molecular de outros pacientes com suspeita clínica e/ou bioquímica de algumas das DLs selecionadas, b) diagnóstico diferencial de um paciente utilizando um dos painéis NGS desenhados e, c) avaliação das amostras de recém-nascidos (obtidas em estudo paralelo) que tiveram resultados inicialmente alterados na triagem neonatal bioquímica para algumas das DLs selecionadas. Resultados: 1) Três painéis foram desenhados, cada um consistindo em dois pools de primers que amplificam as regiões codificantes e 20pb da junção exon-intron. Os painéis A, B e C têm amplitude de cobertura de 97.74, 99.6 e 98.38%, respectivamente. Nesta validação foi possível estabelecer a sensibilidade, especificidade e limitações de cada painel (Artigo 1); 2) foi estabelecida a metodologia para a extração de ADN para seu uso em vários processos moleculares downstream: PCR convencional, Real-Time PCR, PCR-RFLP, MLPA, Sequenciamento de Sanger e NGS na plataforma Ion Torrent PGMTM (Artigo 2); 3) A utilidade clínica dos painéis desenhados foi estabelecida através de: a) Genotipagem de 48 pacientes com suspeita clínica e bioquímica de CLN2 (painel C) (Artigo 4), e de 3 outros casos: 1 caso de Niemann-Pick tipo B (SMPD1) (painel B) e 2 casos de doença de Danon (LAMP2) (painel A) (Artigo 1), b) o diagnóstico diferencial foi realizado através do caso de um paciente com suspeita inicial de Niemann-Pick C que, após a análise molecular, foi diagnosticado com Niemann-Pick tipo B utilizando o painel B, (Artigo 1) c) a aplicabilidade dos painéis de genes para a confirmação diagnóstica em casos de triagem neonatal foi realizada em 2 casos com resultados alterados na triagem neonatal: um caso de Pompe (painel A) e outro de Gaucher (painel B) (Artigo 3 e Anexo I). Conclusão: A abordagem por NGS através do uso de painéis, foi capaz de identificar diferentes alterações genéticas nos genes estudados, incluindo variantes do tipo missense, nonsense, de sítio de splicing, e pequenas indels. Estes painéis oferecem uma estratégia de triagem dos 24 genes associadas às DLs selecionadas. Este trabalho foi inovador ao utilizar o NGS para a análise molecular dos genes associados às DLs no Brasil; foi desenvolvido no Laboratório de Genética e Biologia Molecular do Serviço de Genética Médica do Hospital de Clínicas de Porto Alegre, conhecido como um centro de referência em diagnóstico e pesquisa de DLs, e que atualmente tem os painéis NGS como principal ferramenta de diagnóstico molecular.Introduction: Lysosomal diseases (LDs) are genetic pathologies that, although classified as rare, affect a significant percentage of the population. Many factors make its diagnosis challenging, including variability in phenotype, with few specific clinical signs and symptoms. The development of innovative research tools, highlighting new methods of massive sequencing, would reduce the "diagnostic odyssey" faced by the patient and his family, would provide an earlier diagnosis with better treatment outcome in situations where therapy is available, in addition to adequate genetic counseling. Next-generation sequencing (NGS) technology has clear advantages over conventional sequencing techniques, offering a high diagnostic yield by enabling a comprehensive mutational spectrum to be defined. The NGS allows the sequencing of several genes simultaneously with relatively low overall cost, making gene panels an attractive alternative for genetic screening. This approach is able to detect, in a highly specific manner, missense, nonsense, splicing, and small indels variants, and some large deletions in homozygosis or hemizygosis. However, for this technology to be applicable to clinical practice, a prior validation step is required to determine the critical parameters of analysis from sample processing to data analysis and interpretation. After validation of the methods, it will be possible to evaluate the clinical usefulness of NGS panels for several applications, including their use as a confirmatory method in cases of altered neonatal screening for lysosomal diseases. Objectives: 1) To design and validate an NGS-based strategy for the analysis of 24 genes, included in 3 different panels according to pre-defined parameters, and associated with 22 LDs; 2) To evaluate the quality and efficiency of DNA extracted from dried blood spots (DBS) for its use in NGS; 3) To evaluate the clinical use of NGS for: a) molecular diagnosis, b) differential diagnosis and c) diagnostic confirmation of altered cases in neonatal screening for at least 4 of these conditions (Gaucher, Fabry, Pompe and Niemann-Pick type A / B). Methodology: Descriptive study, convenience sampling, including patients with clinical and biochemical diagnosis of selected LDs. The NGS Ion Torrent Personal Genome Machine (Thermo Fisher Scientific) platform was used for the sequencing of three gene panels, designed using the Ion Ampliseq ™ Designer tool (Thermo Fisher Scientific). For the validation, patients with previous molecular diagnosis by Sanger sequencing were included. For the extraction of DBS DNA, a non-commercial method of phenol-chloroform was evaluated. The clinical utility of NGS was established through: a) molecular diagnosis of a group of patients with suspected type 2 neuronal cercoid lipofuscinosis (CLN2) to establish the disease-associated genotype (TPP1), as well as the molecular diagnosis of other patients with clinical and / or biochemical suspicion of some of the selected LDs, b) differential diagnosis of a patient using one of the designed NGS panels, and c) evaluation of the newborn samples (obtained in a parallel study) that initially had altered results in the neonatal screening for some of the selected LDs. Results: 1) Three panels were designed, each consisting of two pools of primers that amplify the coding regions and 20pb of the exon-intron junction. Panels A, B and C have coverage range of 97.74, 99.6 and 98.38%, respectively. In this validation it was possible to establish the sensitivity, specificity and limitations of each panel (Article 1); 2) the methodology for the extraction of DNA for its use in several downstream molecular processes was established: conventional PCR, Real-Time PCR, PCR-RFLP, MLPA, Sanger Sequencing and NGS in the Ion Torrent PGMTM platform; 3) The clinical use of the panels was established through: a) Genotyping of 48 patients with clinical and biochemical CLN2 (panel C) (Article 4), and of 3 other cases: 1 case of Niemann-Pick type B ( SMPD1) (panel B) and 2 cases of Danon's disease (LAMP2) (panel A) (Article 1), b) he differential diagnosis was made through the case of a patient with initial suspicion of Niemann-Pick C who, after molecular analysis, was diagnosed with Niemann-Pick type B using panel B (Article 1), c) the applicability of the gene panels for diagnostic confirmation in cases of neonatal screening was performed in 2 cases with altered results in neonatal screening: one case of Pompe (panel A) and another case of Gaucher (panel B) (Article 3 and Annex I) . Conclusion: The NGS approach using gene panels was able to identify different genetic alterations in the genes studied, including missense, nonsense, splicing variants, and small indels. These panels offer a screening strategy for the 24 genes associated with the selected LDs. This work was innovative by using NGS for the molecular analysis of genes associated with LDs in Brazil; it was developed in the Laboratory of Genetics and Molecular Biology of the Medical Genetics Service of the Hospital de Clinicas de Porto Alegre, known as a reference center in diagnosis and research of LDs, and that currently has the NGS panels as the main tool of molecular diagnosis

Scalable Computational Frameworks for Next-Generation Sequencing Analysis and Gene Set Integration

The rapid growth in biomedical research has generated vast amounts of data, including genomic, molecular, imaging, and clinical information from humans and other species. Leveraging this data is essential for groundbreaking scientific discoveries and a deeper understanding of health and disease across different species. However, the complexity and volume of these datasets present significant computational challenges, limiting their potential.This dissertation addresses two key challenges in biomedical data analysis: the efficient evaluation of sequencing data and the effective management and analysis of gene sets. By focusing on these areas, we develop innovative computational methods that enable the rapid, scalable, and accurate processing of large-scale biomedical data. For sequencing data, we create algorithms that enhance the speed and precision of data evaluation, making it feasible to manage the increasing volume of sequences generated by modern technologies. For gene sets, we devise tools for their efficient management and analysis, allowing researchers to draw meaningful insights from complex genetic information. Through this research, we aim to contribute to the development of new analytical tools and methods, ultimately supporting the advancement of precision medicine and personalized healthcare for both human and veterinary applications

A new approach to Congenital Dyserythropoietic Anemias: toward a better definition of molecular mechanisms

Hereditary hemolytic anemias (HHAs) embrace a highly heterogeneous group of chronic disorders with a highly variable clinical picture. HHA encompass (1) hyporegenerative anemias (HAs), as congenital dyserythropoietic anemias (CDAs); (2) hemolytic anemias due to red cell membrane defects (HAMDs), as hereditary spherocytosis (HS) and hereditary stomatocytosis (HST). Although the workflow to diagnose these conditions is a normal clinical practice, differential diagnosis, classification, and patient stratification among HHAs are often very difficult. Beyond achieving a definitive diagnosis, knowing the genetic basis of these patients can be valuable also for guiding treatment. Next generation sequencing (NGS) refers to non-Sanger-based high-throughput DNA sequencing technologies. This technology plays a major role either in disease gene discovery or in clinical use for establishing a genetic diagnosis. Particularly, the major current application of NGS in diagnostics is through design of disease specific panel, named targeted (t)-NGS, in which a selected fraction of genes is sequenced. The primary aim of our study was the development of a fast, accurate, reliable and cost effective diagnostic/prognostic tool for HHAs based on t-NGS. In order to assess the reliability of this approach we created a t-NGS gene panel, named RedPlex, composed by 34 loci causative or candidates of HHAs. In silico design was performed by Agilent SureDesign web tool. For each locus, all coding regions, 5’ and 3’UTRs, and 100 bp flanking splice junctions were included. Sequence length was set at 150×2 nucleotides, and the predicted target size amounted to 538 regions (239.764 kb). Targeted enrichment was performed on 32 patients from 27 unrelated families by HaloPlex Target Enrichment System. High-throughput sequencing was performed by Illumina NextSeq 500. SureCall software was used for bioinformatic and computational analyses. RedPlex panel showed high sensitivity and specificity. It was able to capture at least 99.4% of 538 target regions with high and uniform coverage. We were able to obtain a conclusive diagnosis in approximately 72% of cases. In addition, a lot of patients (39%) showed multiple disease-associated variants suggesting complex inheritance. Indeed, t-NGS approach also allows the identification of “polygenic” genotypes, which may account for the phenotypic variability among HHA patients. Thus, the secondary aim of this project was the study of the interaction between mutated genes in HHA patients. We particularly focused on the functional interaction between two CDA-related genes, GATA1 and SEC23B. The demonstration of the direct interaction of GATA1 transcription factor on the SEC23B promoter provided also an explanation of the variability of phenotypes GATA1-related by means of the crosstalk of this gene with its target SEC23B

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

SITC cancer immunotherapy resource document: a compass in the land of biomarker discovery.

Since the publication of the Society for Immunotherapy of Cancer\u27s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients

Current Utility and Future Applications of ctDNA in Colorectal Cancer

Circulating tumour DNA (ctDNA) shows promise as a minimally invasive biomarker with a myriad of emerging applications including early detection and diagnosis, monitoring of disease and treatment efficacy, and identification of actionable alterations to guide treatment. The potential utility of ctDNA in colorectal carcinoma (CRC) is of particular interest given the limitations of current radiographic imaging and blood-based tumour markers in detecting disease and evaluating therapeutic benefit. While ctDNA has yet to demonstrate clinical utility in CRC, a growing body of research highlights the potential of these novel biomarkers. This chapter provides an overview of the current evidence for employing ctDNA in CRC as well as previewing the future directions that these exciting technologies may take

Development and application of methodologies and infrastructures for cancer genome analysis within Personalized Medicine

[eng] Next-generation sequencing (NGS) has revolutionized biomedical sciences, especially in the area of cancer. It has nourished genomic research with extensive collections of sequenced genomes that are investigated to untangle the molecular bases of disease, as well as to identify potential targets for the design of new treatments. To exploit all this information, several initiatives have emerged worldwide, among which the Pan-Cancer project of the ICGC (International Cancer Genome Consortium) stands out. This project has jointly analyzed thousands of tumor genomes of different cancer types in order to elucidate the molecular bases of the origin and progression of cancer. To accomplish this task, new emerging technologies, including virtualization systems such as virtual machines or software containers, were used and had to be adapted to various computing centers. The portability of this system to the supercomputing infrastructure of the BSC (Barcelona Supercomputing Center) has been carried out during the first phase of the thesis. In parallel, other projects promote the application of genomics discoveries into the clinics. This is the case of MedPerCan, a national initiative to design a pilot project for the implementation of personalized medicine in oncology in Catalonia. In this context, we have centered our efforts on the methodological side, focusing on the detection and characterization of somatic variants in tumors. This step is a challenging action, due to the heterogeneity of the different methods, and an essential part, as it lays at the basis of all downstream analyses. On top of the methodological section of the thesis, we got into the biological interpretation of the results to study the evolution of chronic lymphocytic leukemia (CLL) in a close collaboration with the group of Dr. Elías Campo from the Hospital Clínic/IDIBAPS. In the first study, we have focused on the Richter transformation (RT), a transformation of CLL into a high-grade lymphoma that leads to a very poor prognosis and with unmet clinical needs. We found that RT has greater genomic, epigenomic and transcriptomic complexity than CLL. Its genome may reflect the imprint of therapies that the patients received prior to RT, indicating the presence of cells exposed to these mutagenic treatments which later expand giving rise to the clinical manifestation of the disease. Multiple NGS- based techniques, including whole-genome sequencing and single-cell DNA and RNA sequencing, among others, confirmed the pre-existence of cells with the RT characteristics years before their manifestation, up to the time of CLL diagnosis. The transcriptomic profile of RT is remarkably different from that of CLL. Of particular importance is the overexpression of the OXPHOS pathway, which could be used as a therapeutic vulnerability. Finally, in a second study, the analysis of a case of CLL in a young adult, based on whole genome and single-cell sequencing at different times of the disease, revealed that the founder clone of CLL did not present any somatic driver mutations and was characterized by germline variants in ATM, suggesting its role in the origin of the disease, and highlighting the possible contribution of germline variants or other non-genetic mechanisms in the initiation of CLL