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Next Generation Sequencing in Clinical Diagnostics

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A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (25 November 2023) | Viewed by 34921

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Special Issue Editors

Dr. Pawel Mroz

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Guest Editor

Department of Laboratory Medicine and Pathology, University of Minnesota System, Minneapolis, USA
Interests: digital droplet PCR; pharmacogenomics; SMRT sequencing; minimal residual disease

Dr. Bryan L. Betz

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Guest Editor

Department of Pathology, University of Michigan, Ann Arbor, USA
Interests: emerging technologies; clinical molecular diagnostics; cancer molecular genetics

Dr. Charles Billington

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Guest Editor

Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA
Interests: variable phenotypes; genetic conditions; brain and facial malformations; urea cycle disorders and maple syrup urine disease

Special Issue Information

Dear Colleagues,

Over the last decade, Next Generation Sequencing (NGS), also referred to as Massively Parallel Sequencing, has become a mainstream sequencing technology that has dramatically changed clinical and research capabilities. The constant evolution of NGS platforms, from amplicon-based targeted sequencing to whole genome and transcriptome sequencing, from Ion Torrent and Illumina chemistries to Pacific Biosciences and Oxford Nanopore long read NGS, has resulted in a continuously growing number of clinical applications offering invaluable individualized data to help deliver the promise of personalized medicine. Due to a dramatic reduction in costs, and improvements to library preparation methods, sequencing chemistries, and analysis pipelines, NGS has been widely implemented as a standard of care to detect clinically actionable somatic and germline variants. In the process, NGS has become an essential tool in molecular diagnosis, prognosis, targeted therapy, and for monitoring disease recurrence and evolution. It has been successfully applied to a growing list of clinical applications including oncology, infectious disease, prenatal diagnosis, carrier detection, medical genetics, pharmacogenetics, and histocompatibility typing for transplant.

This Special Issue, entitled “Next Generation Sequencing in Clinical Diagnostics”, aims to present and summarise the enormous progress that has occurred in the clinical implementation of NGS testing as well as offer an outlook to the future of NGS clinical applications. We invite original research and reviews describing the development, implementation, application and advances of NGS testing in the clinical setting.

Dr. Pawel Mroz
Dr. Bryan L. Betz
Dr. Charles Billington
Guest Editors

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Keywords

next generation sequencing
oncology
germline
molecular diagnostics
clinical applications

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Published Papers (9 papers)

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Research

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10 pages, 1115 KiB

Open AccessArticle

Efficient Lung Cancer Molecular Diagnostics by Combining Next Generation Sequencing with Reflex Idylla Genefusion Assay Testing

by Dingani Nkosi, Giby V. George, Huijie Liu, Meghan Buldo, Moises J. Velez and Zoltán N. Oltvai

Genes 2023, 14(8), 1551; https://doi.org/10.3390/genes14081551 - 28 Jul 2023

Cited by 1 | Viewed by 1717

Abstract

Molecular diagnostics for lung cancer is a well-established standard of care, but how to use the available diagnostic tools for optimal and cost-effective patient care remains unresolved. Here, we show that DNA-only, small gene next-generation sequencing (sNGS) panels (<50 genes) combined with ultra-rapid reflex testing for common fusion transcripts using the Idylla Genefusion assay provide a cost-effective and sufficiently comprehensive testing modality for the majority of lung cancer cases. We also demonstrate the need for additional reflex testing capability on larger DNA and fusion panels for a small subset of lung cancers bearing rare single-nucleotide variants, indels and fusion transcripts and secondary, post-treatment resistance mutations. A similar testing workflow could be adopted for other solid tumor types for which extensive gene/fusion variant profiles are available both in the treatment-naïve and post-therapy settings. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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9 pages, 1642 KiB

Open AccessCommunication

Targeted Panel Sequencing Identifies an Intronic c.5225-3C>G Variant of the FBN1 Gene Causing Sporadic Marfan Syndrome with Annuloaortic Ectasia

by Kyung Hwa Kim, Tae Yun Kim, Soon Jin Kim, Yong Gon Cho, Joonhong Park and Woori Jang

Genes 2022, 13(11), 2108; https://doi.org/10.3390/genes13112108 - 13 Nov 2022

Viewed by 1828

Abstract

Marfan syndrome (MFS) is a hereditary connective tissue disease whose clinical severity varies widely. Mutations of the FBN1 gene encoding fibrillin-1 are the most common genetic cause of Marfanoid habitus; however, about 10% of MFS patients are unaware of their genetic defects. Herein, we report a Korean patient with MFS and annuloaortic ectasia caused by an intronic c.5225-3C>G variant of the FBN1 gene identified by targeted panel sequencing. The reverse transcription analysis of FBN1 revealed that the intron 43 sequence from positions c.5297-1516 to c.5297-1 was retained at the coding sequence as a consequence of the c.5225-3C>G variant enhancing a cryptic splice acceptor site (c.5297-1518_5297-1517AG) in intron 43. The retained sequence of the part of intron 43 caused the same effect as insertion mutation (NM_000138.5:c.5297_c.5298ins5297-1516_5297-1), resulting in a frameshift mutation resulting in p.Ile1767Trpfs*3. The patient underwent an urgent modified Bentall operation with a 29 mm mechanical valve for annuloaortic ectasia and severe aortic valve regurgitation. This report emphasizes the need for functional investigations into the diagnostic workflows of certain diseases or gene panels with suspected high rates of intronic variants and potential pathogenic effects. Hence, further descriptions of individuals with intronic variants causing alternative splicing expected to have pathogenic effects at different transcript levels are crucial for improving our understanding. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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11 pages, 1561 KiB

Open AccessCommunication

Gene Panel Sequencing Identifies a Novel RYR1 p.Ser2300Pro Variant as Candidate for Malignant Hyperthermia with Multi-Minicore Myopathy

by Young Jae Moon, Joonhong Park, Jung Ryul Kim, Seung Yeob Lee, Jaehyeon Lee, Yong Gon Cho and Dal Sik Kim

Genes 2022, 13(10), 1726; https://doi.org/10.3390/genes13101726 - 26 Sep 2022

Cited by 1 | Viewed by 2194

Abstract

Malignant hyperthermia (MH), a rare autosomal dominant pharmacogenetic disorder of skeletal muscle calcium regulation, is triggered by sevoflurane in susceptible individuals. We report a Korean having MH with multi-minicore myopathy functionally supported by RYR1-mediated intracellular Ca²⁺ release testing in B lymphocytes. A 14-year-old boy was admitted for the evaluation of progressive torticollis accompanied by cervicothoracic scoliosis. During the preoperative drape of the patient for the release of the sternocleidomastoid muscle under general anesthesia, his wrist and ankle were observed to have severe flexion contracture. The body temperature was 37.1 °C. To treat MH, the patient was administered a bolus of dantrolene intravenously (1.5 mg/kg) and sodium bicarbonate. After a few minutes, muscle rigidity, tachycardia, and EtCO2 all resolved. Next-generation panel sequencing for hereditary myopathy identified a novel RYR1 heterozygous missense variant (NM_000540.2: c.6898T > C; p.Ser2300Pro), which mapped to the MH2 domain of the protein, a hot spot for MH mutations. Ex vivo RYR1-mediated intracellular Ca²⁺ release testing in B lymphocytes showed hypersensitive Ca²⁺ responses to isoflurane and caffeine, resulting in an abnormal Ca²⁺ release only in the proband, not in his family members. Our findings expand the clinical and pathological spectra of information associated with MH with multi-minicore myopathy. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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11 pages, 1681 KiB

Open AccessEditor’s ChoiceArticle

Utility of Select Gene Mutation Detection in Tumors by the Idylla Rapid Multiplex PCR Platform in Comparison to Next-Generation Sequencing

by Dingani Nkosi, Vektra L. Casler, Chauncey R. Syposs and Zoltán N. Oltvai

Genes 2022, 13(5), 799; https://doi.org/10.3390/genes13050799 - 29 Apr 2022

Cited by 5 | Viewed by 2522

Abstract

Testing of tumors by next generation sequencing (NGS) is impacted by relatively long turnaround times and a need for highly trained personnel. Recently, Idylla oncology assays were introduced to test for BRAF, EGFR, KRAS, and NRAS common hotspot mutations that do not require specialized trained personnel. Moreover, the interpretation of results is fully automated, with rapid turnaround time. Though Idylla testing and NGS have been shown to have high concordance in identifying EGFR, BRAF, KRAS, and NRAS hotspot mutations, there is limited experience on optimal ways the Idylla system can be used in routine practice. We retrospectively evaluated all cases with EGFR, BRAF, KRAS, or NRAS mutations identified in clinical specimens sequenced on two different NGS panels at the University of Rochester Medical Center (URMC) molecular diagnostics laboratory between July 2020 and July 2021 and assessed if these mutations would be detected by the Idylla cartridges if used. We found that the Idylla system could accurately identify Tier 1 or 2 actionable genomic alterations in select associated disease pathologies if used. Yet, in a minority of cases, we would have been unable to detect NGS-identified pathogenic mutations due to their absence on the Idylla panels. We derived algorithmic practice guidelines for the use of the Idylla cartridges. Overall, Idylla molecular testing could be implemented either as a first-line standalone diagnostic tool in select indications or for orthogonal confirmation of uncertain results. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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12 pages, 3046 KiB

Open AccessArticle

Technological Advances: CEBPA and FLT3 Internal Tandem Duplication Mutations Can be Reliably Detected by Next Generation Sequencing

by Ratilal Akabari, Dahui Qin and Mohammad Hussaini

Genes 2022, 13(4), 630; https://doi.org/10.3390/genes13040630 - 1 Apr 2022

Cited by 2 | Viewed by 3694

Abstract

Background: The detection of CEBPA and FLT3 mutations by next generation sequencing (NGS) is challenging due to high GC content and Internal Tandem Duplications (ITDs). Recent advances have been made to surmount these challenges. In this study, we compare three commercial kits and evaluate the performance of these more advanced hybrid-capture and AMP-chemistry based methods. Methods: Amplicon-based TSM 54-Gene Panel (Illumina) was evaluated against hybridization-capture SOPHiA Genetics MSP, OGT SureSeq, and AMP chemistry-based VariantPlex (Archer) for wet-lab workflow and data-analysis pipelines. Standard kit directions and commercial analysis pipelines were followed. Seven CEBPA and 10 FLT3-positive cases were identified that previously were missed on an amplicon NGS assay. The average reads, coverage uniformity, and the detection of CEBPA or FLT3 mutations were compared. Results: All three panels detected all 10 CEBPA mutations and all 10 FLT3 ITDs with 100% sensitivity. In addition, there was high concordance (100%) between all three panels detecting 47/47 confirmed variants in a set of core myeloid genes. Conclusions: The results show that the NGS assays are now able to reliably detect CEBPA mutations and FLT3 ITDs. These assays may allow foregoing additional orthogonal testing for CEBPA and FLT3. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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7 pages, 935 KiB

Open AccessCommunication

Identification of a Cancer-Predisposing Germline POT1 p.Ile49Metfs*7 Variant by Targeted Sequencing of a Splenic Marginal Zone Lymphoma

by Audrey N. Jajosky, Anna L. Mitchell, Mahmut Akgul, Shashirekha Shetty, Jennifer M. Yoest, Stanton L. Gerson, Navid Sadri and Kwadwo A. Oduro, Jr.

Genes 2022, 13(4), 591; https://doi.org/10.3390/genes13040591 - 26 Mar 2022

Cited by 1 | Viewed by 2869

Abstract

Germline disruptive variants in Protection of Telomeres 1 (POT1) predispose to a wide variety of cancers, including melanoma, chronic lymphocytic leukemia (CLL), Hodgkin lymphoma, myeloproliferative neoplasms, and glioma. We report the first case of splenic marginal zone lymphoma (SMZL) arising in a patient with a germline POT1 variant: a 65-year-old male with an extensive history of cancer, including melanoma and papillary thyroid carcinoma, who presented with circulating atypical lymphocytosis. Bone marrow biopsy revealed 20% involvement by a CD5⁻CD10⁻ B-cell lymphoma that was difficult to classify. During the clinical workup of his low-grade lymphoma, targeted next-generation sequencing (NGS) identified POT1 p.I49Mfs*7 (NM_015450:c. 147delT) at a variant allele frequency (VAF) of 51%. NGS of skin fibroblasts confirmed the POT1 variant was germline. This likely pathogenic POT1 loss-of-function variant has only been reported once before as a germline variant in a patient with glioma and likely represents one of the most deleterious germline POT1 variants ever linked to familial cancer. The spectrum of cancers associated with germline pathogenic POT1 variants (i.e., autosomal dominant POT1 tumor predisposition syndrome) should potentially be expanded to include SMZL, a disease often associated with the loss of chromosome 7q: the location of the POT1 genetic locus (7q31.33). Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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8 pages, 2257 KiB

Open AccessArticle

Visualization of the Effect of Assay Size on the Error Profile of Tumor Mutational Burden Measurement

by Nathanael G. Bailey

Genes 2022, 13(3), 432; https://doi.org/10.3390/genes13030432 - 26 Feb 2022

Cited by 1 | Viewed by 1886

Abstract

Tumor mutational burden (TMB) refers to the number of somatic mutations in a tumor per megabase and is a biomarker for response to immune checkpoint inhibitor therapy. Immune checkpoint inhibitors are currently approved for tumors with TMB greater than or equal to 10 mutations/megabase. Many laboratories are currently reporting TMB values based upon targeted resequencing panels with limited genomic coverage. Due to sampling variation, this leads to significant uncertainty in the assay’s TMB result, particularly at relatively low TMB levels near the 10 mutation per megabase therapeutic threshold. In order to allow clinicians and laboratorians to explore this uncertainty, we built a novel web application that allows a user to view the potential error of a TMB result given the sequencing panel size. This application also allows the user to explore the effect of incorporating knowledge of a specific tumor type’s typical TMB distribution on the error profile of the TMB result. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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Review

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24 pages, 1345 KiB

Open AccessEditor’s ChoiceReview

Next Generation and Other Sequencing Technologies in Diagnostic Microbiology and Infectious Diseases

by Evann E. Hilt and Patricia Ferrieri

Genes 2022, 13(9), 1566; https://doi.org/10.3390/genes13091566 - 31 Aug 2022

Cited by 57 | Viewed by 14524

Abstract

Next-generation sequencing (NGS) technologies have become increasingly available for use in the clinical microbiology diagnostic environment. There are three main applications of these technologies in the clinical microbiology laboratory: whole genome sequencing (WGS), targeted metagenomics sequencing and shotgun metagenomics sequencing. These applications are being utilized for initial identification of pathogenic organisms, the detection of antimicrobial resistance mechanisms and for epidemiologic tracking of organisms within and outside hospital systems. In this review, we analyze these three applications and provide a comprehensive summary of how these applications are currently being used in public health, basic research, and clinical microbiology laboratory environments. In the public health arena, WGS is being used to identify and epidemiologically track food borne outbreaks and disease surveillance. In clinical hospital systems, WGS is used to identify multi-drug-resistant nosocomial infections and track the transmission of these organisms. In addition, we examine how metagenomics sequencing approaches (targeted and shotgun) are being used to circumvent the traditional and biased microbiology culture methods to identify potential pathogens directly from specimens. We also expand on the important factors to consider when implementing these technologies, and what is possible for these technologies in infectious disease diagnosis in the next 5 years. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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Other

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7 pages, 1039 KiB

Open AccessCase Report

Live Birth of a Healthy Child in a Couple with Identical mtDNA Carrying a Pathogenic c.471_477delTTTAAAAinsG Variant in the MOCS2 Gene

by Maria Tofilo, Natalia Voronova, Leila Nigmatullina, Elena Kuznetsova, Valeria Timonina, Bogdan Efimenko, Oybek Turgunkhujaev, Svetlana Avdeichik, Muhammad Ansar, Konstantin Popadin, Anastasia Kirillova and Ilya Mazunin

Genes 2023, 14(3), 720; https://doi.org/10.3390/genes14030720 - 15 Mar 2023

Viewed by 1777

Abstract

Molybdenum cofactor deficiency type B (MOCODB; #252160) is an autosomal recessive metabolic disorder that has only been described in 37 affected patients. In this report, we describe the presence of an in-frame homozygous variant (c.471_477delTTTAAAAinsG) in the MOCS2 gene in an affected child, diagnosed with Ohtahara syndrome according to the clinical manifestations. The analysis of the three-dimensional structure of the protein and the amino acid substitutions suggested the pathogenicity of this mutation. To prevent transmitting this mutation to the next generation, we used preimplantation genetic testing for the monogenic disorders (PGT-M) protocol to select MOCS2 gene mutant-free embryos for transfer in an in vitro fertilization (IVF) program. As a result, a healthy child was born. Interestingly, both parents of the proband shared an identical mitochondrial (mt) DNA control region, assuming their close relationship and thus suggesting that both copies of the nuclear rare variant c.471_477delTTTAAAAinsG may have been transmitted from the same female ancestor. Our estimation of the a priori probability of meeting individuals with the same mtDNA haplotype confirms the assumption of a possible distant maternal relationship among the proband’s direct relatives. Full article

(This article belongs to the Special Issue Next Generation Sequencing in Clinical Diagnostics)

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