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Next-Generation Sequencing (NGS) Technique and Personalized Medicine
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Next-Generation Sequencing (NGS) Technique and Personalized Medicine

A special issue of Current Issues in Molecular Biology (ISSN 1467-3045). This special issue belongs to the section "Molecular Medicine".

Deadline for manuscript submissions: closed (31 May 2023) | Viewed by 31953

Special Issue Editors

Dr. Chiara Rosso

E-Mail Website
Guest Editor
Department of Medical Sciences, University of Turin, 10126 Turin, Italy
Interests: non-alcoholic fatty liver disease (NAFLD); non-invasive biomarkers for risk stratification in NAFLD; pathophysiology of insulin resistance; effect of diet and lifestyle intervention on insulin resistance and NAFLD severity
Special Issues, Collections and Topics in MDPI journals
Dr. Antonio Francavilla

E-Mail Website
Guest Editor
Italian Institute for Genomic Medicine (IIGM), c/o IRCCS Candiolo, 10060 Candiolo TO, Italy
Interests: colorectal cancer; celiac disease; nutrition; non-invasive biomarkers; next-generation sequencing; non-coding RNAs; gut microbiota; multi omics

Special Issue Information

Dear Colleagues,

Next-generation sequencing (NGS) technologies enable the sequencing of multiple genes at a very high throughput and with relatively low cost. The NGS approach has led to a significant step forward in the unbiased transcriptomic analysis of mRNAs, small RNAs, and non-coding RNAs; genome-wide methylation assays; and chromatin immunoprecipitation assays. Furthermore, it has made a major contribution to the personalized medicine field, since NGS enables the detection of both somatic and germline mutations, which are implicated in the onset and progression of various diseases, including cancers. The aim of this Special Issue is to focus on the clinical application of NGS, from molecular alterations to therapeutic approaches and personalized therapy. Authors are invited to submit original papers on the application of NGS in different clinical settings as well as reviews describing the state of the art on this topic.

Dr. Chiara Rosso
Dr. Antonio Francavilla
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Current Issues in Molecular Biology is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2200 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • next-generation sequencing
  • personalized medicine
  • molecular genetic diagnostics
  • cancer genomics

Published Papers (16 papers)

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Research

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15 pages, 3022 KiB  
Article
Characterization of Pulmonary Bacteriobiota in Critically Ill Patients in Southern Peru through Next-Generation Sequencing Technology
by Katherine Quispe-Medina, Jani Pacheco-Aranibar, Angel Mamani-Ruelas, Carlos Gamez-Bernabé, Rosemary Zapana-Begazo, Ivan Paz-Aliaga, Jose Villanueva Salas and Julio C. Bernabé-Ortiz
Curr. Issues Mol. Biol. 2023, 45(12), 10041-10055; https://doi.org/10.3390/cimb45120627 - 13 Dec 2023
Viewed by 950
Abstract
Sequence variation in the 16S gene is widely used to characterize diverse microbial communities. This was the first pilot study carried out in our region where the pulmonary microbiota of critically ill patients was investigated and analyzed, with the aim of finding a [...] Read more.
Sequence variation in the 16S gene is widely used to characterize diverse microbial communities. This was the first pilot study carried out in our region where the pulmonary microbiota of critically ill patients was investigated and analyzed, with the aim of finding a specific profile for these patients that can be used as a diagnostic marker. An study of critical patients mechanically ventilated for non-respiratory indications, in a polyvalent intensive care unit, was carried out; samplee were extracted by endotracheal aspiration and subsequently the microbiota was characterized through Next-Generation Sequencing Technology (NGS). The predominant phyla among the critically ill patients were Proteobacteria, Firmicutes and Bacteroidata. In the surviving patients group, the predominant phyla were Proteobacteria, Bacteroidata and Firmicutes, in the group of deceased patients thy were Firmicutes, Proteobacteria, and Bacteroidata. We found a decrease in commensal bacteria in deceased patients and a progressive increase in in-hospital germs. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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13 pages, 757 KiB  
Article
Development and Evaluation of a New qPCR Assay for the Detection of Mycoplasma in Cell Cultures
by José A. Carrillo-Ávila, Amanda de la Fuente, Rocío Aguilar-Quesada, Gertrudis Ligero, Juan Manuel del Río-Ortiz and Purificación Catalina
Curr. Issues Mol. Biol. 2023, 45(8), 6903-6915; https://doi.org/10.3390/cimb45080435 - 18 Aug 2023
Cited by 2 | Viewed by 1902
Abstract
In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact [...] Read more.
In recent years, cell culture has become an important tool not only in research laboratories, but also in diagnostic and biotechnological development laboratories. Mycoplasma contamination is present in up to 35% of cell cultures used in research and in cell therapies. This fact represents a significant problem since such contamination can cause disastrous effects on eukaryotic cells by altering their cellular parameters, which, in turn, can lead to unreliable experimental results. For this reason, it is mandatory to carry out continuous testing for the presence of Mycoplasma in cell culture and the development of appropriate methodologies for this purpose. An ideal detection methodology should be fast, sensitive, and reliable. In this study, we propose an alternative detection method based on real-time PCR in conjunction with a novel combination of primers and probes that have been improved to increase their efficiency. The new PCR method demonstrates 100% sensitivity and specificity results in the detection of common Mycoplasma species that contaminate cell cultures. Whilst 11 of 45 tested supernatants were positive for Mycoplasma (24.4%) using the new PCR method (corresponding to 5 of the 14 lines tested (35.71%)), only 10 of 45 supernatants showed positive results with the commercial Venor®GeM qEP and Plasmotest® kit. In addition, the new PCR method exhibits a high capacity to detect less-frequent Mycoplasma species, such as those related to the M. mycoides cluster. The use of an alternative Mycoplasma-detection method in cell culture labs can guarantee the detection of Mycoplasma contamination, especially in cases when dubious results are recorded. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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12 pages, 563 KiB  
Communication
The Spectrum of Germline Nucleotide Variants in Gastric Cancer Patients in the Kyrgyz Republic
by Airat Bilyalov, Sergey Nikolaev, Anastasiia Danishevich, Igor Khatkov, Komron Makhmudov, Zhainagul Isakova, Nurbek Bakirov, Ernis Omurbaev, Alena Osipova, Ramaldan Ramaldanov, Elena Shagimardanova, Andrey Kiyasov, Oleg Gusev and Natalia Bodunova
Curr. Issues Mol. Biol. 2023, 45(8), 6383-6394; https://doi.org/10.3390/cimb45080403 - 31 Jul 2023
Viewed by 1351
Abstract
Gastric cancer is a major challenge in modern oncology due to its high detection rate and prevalence. While sporadic cases make up the majority of gastric cancer, hereditary gastric cancer is caused by germline mutations in several genes linked to different syndromes. Thus, [...] Read more.
Gastric cancer is a major challenge in modern oncology due to its high detection rate and prevalence. While sporadic cases make up the majority of gastric cancer, hereditary gastric cancer is caused by germline mutations in several genes linked to different syndromes. Thus, identifying hereditary forms of gastric cancer is considered crucial globally. A survey study using NGS-based analysis was conducted to determine the frequency of different types of hereditary gastric cancer in the yet-unstudied Kyrgyz population. The study cohort included 113 patients with diagnosed gastric cancer from Kyrgyzstan. The age of patients was 57.6 ± 8.9. Next-generation sequencing analysis of genomic DNA was performed using a custom Roche NimbleGen enrichment panel. The results showed that 6.2% (7/113) of the patients had pathogenic or likely pathogenic genetic variants. Additionally, 3.5% (4/113) of the patients carried heterozygous pathogenic/likely pathogenic variants in high penetrance genes, such as TP53, POLD1, RET, and BRCA2. Moreover, 2.7% (3/113) of the patients carried heterozygous mutations in genes linked to autosomal recessive conditions, specifically PALB2, FANCA, and FANCD2. We have not identified any genetic variants in hereditary GC-associated genes: CDH1, STK11, SMAD4, BMPRIA, APC, MLH1, and others. Our study included patients with sporadic features of GC. The use of recognized criteria (NCCN, Gastric Cancer, Version 2.2022) would increase the number of identified genetic variants in hereditary GC-associated genes. Further research is required to determine the clinical relevance of the genetic variants identified in the current study. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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12 pages, 1855 KiB  
Article
Whole-Exome Sequencing Identified Two Novel Pathogenic Mutations in the PTCH1 Gene in BCNS
by Margit Pál, Éva Vetró, Nikoletta Nagy, Dóra Nagy, Emese Horváth, Barbara Anna Bokor, Anita Varga, László Seres, Judit Oláh, József Piffkó and Márta Széll
Curr. Issues Mol. Biol. 2023, 45(7), 5293-5304; https://doi.org/10.3390/cimb45070336 - 24 Jun 2023
Cited by 1 | Viewed by 1229
Abstract
Basal cell nevus syndrome (BCNS, OMIM 109400) is a familial cancer syndrome characterized by the development of numerous basal cell cancers and various other developmental abnormalities, including epidermal cysts of the skin, calcified dural folds, keratocysts of the jaw, palmar and plantar pits, [...] Read more.
Basal cell nevus syndrome (BCNS, OMIM 109400) is a familial cancer syndrome characterized by the development of numerous basal cell cancers and various other developmental abnormalities, including epidermal cysts of the skin, calcified dural folds, keratocysts of the jaw, palmar and plantar pits, ovarian fibromas, medulloblastomas, lymphomesenteric cysts, and fetal rhabdomyomas. BCNS shows autosomal dominant inheritance and is caused by mutations in the patched 1 (PTCH1) gene and the suppressor of the fused homolog (SUFU) gene. In a few cases, variants of patched 2 (PTCH2) have been found in patients who met the criteria for BCNS. In an investigation of 11 Hungarian families who fulfilled the diagnostic criteria for BCNS, whole-exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) identified two novel pathogenic variants (c.2994C>A; p.Cys998Ter and c.814_818del; p.Asn272SerfsTer11), one recently identified variant (c.1737_1745del p.Val580_Val582del), and three recurrent disease-causing variants of the PTCH1 gene with a diagnosis rate of 63.6%. Disease-causing variants were not found for the SUFU and PTCH2 genes. These applied methods could not fully elucidate the genetic background of all the BCNS cases that we investigated. To uncover the missing heritability of BCNS, whole-genome sequencing or an epigenetic approach might be considered in the future. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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12 pages, 1581 KiB  
Article
Identifying Genomic Signatures of Positive Selection to Predict Protective Genomic Loci in the Cohort of Lithuanian Clean-Up Workers of the Chornobyl Nuclear Disaster
by Gabrielė Žukauskaitė, Ingrida Domarkienė, Aušra Matulevičienė, Svetlana Dauengauer-Kirlienė, Vaidutis Kučinskas and Laima Ambrozaitytė
Curr. Issues Mol. Biol. 2023, 45(4), 2972-2983; https://doi.org/10.3390/cimb45040195 - 03 Apr 2023
Viewed by 1225
Abstract
Some people resist or recover from health challenges better than others. We studied Lithuanian clean-up workers of the Chornobyl nuclear disaster (LCWC) who worked in the harshest conditions and, despite high ionising radiation doses as well as other factors, continue ageing relatively healthily. [...] Read more.
Some people resist or recover from health challenges better than others. We studied Lithuanian clean-up workers of the Chornobyl nuclear disaster (LCWC) who worked in the harshest conditions and, despite high ionising radiation doses as well as other factors, continue ageing relatively healthily. Thus, we hypothesised that there might be individual features encoded by the genome which act protectively for better adaptiveness and health that depend on unique positive selection signatures. Whole-genome sequencing was performed for 40 LCWC and a control group composed of 25 men from the general Lithuanian population (LTU). Selective sweep analysis was performed to identify genomic regions which may be under recent positive selection and determine better adaptiveness. Twenty-two autosomal loci with the highest positive selection signature values were identified. Most important, unique loci under positive selection have been identified in the genomes of the LCWC, which may influence the survival and adaptive qualities to extreme conditions, and the disaster itself. Characterising these loci provide a better understanding of the interaction between ongoing microevolutionary processes, multifactorial traits, and diseases. Studying unique groups of disease-resistant individuals could help create new insights for better, more individualised, disease diagnostics and prevention strategies. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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7 pages, 800 KiB  
Communication
HR Gene Variants Identified in Mexican Patients with Alopecia Areata
by Andrés Ortiz-Ramírez, María Cristina Hernández-Jiménez, Iliana Berenice Guardiola-Avila, Erick de Jesús De Luna-Santillana, Amanda Alejandra Oliva-Hernández, María Lourdes Altamirano-García and Karina Janett Juárez-Rendón
Curr. Issues Mol. Biol. 2023, 45(4), 2965-2971; https://doi.org/10.3390/cimb45040194 - 03 Apr 2023
Cited by 1 | Viewed by 1069
Abstract
Alopecia Areata (AA) is a multifactorial, dermatological disease characterized by non-scarring hair loss. Alterations in candidate genes, such as HR (Hairless), could represent a risk factor for its development. The aim of this study was to search for and analyze variants in exons [...] Read more.
Alopecia Areata (AA) is a multifactorial, dermatological disease characterized by non-scarring hair loss. Alterations in candidate genes, such as HR (Hairless), could represent a risk factor for its development. The aim of this study was to search for and analyze variants in exons 3, 15 and 17 of the HR gene in Mexican patients with AA. A total of 30 samples from both AA patients and healthy donors were analyzed in this study. Exons were amplified and sequenced using the Sanger method. Descriptive statistics and χ2 tests were used in the analysis of clinical–demographic characteristics and the comparison of allelic/genotypical frequencies between groups, respectively. The effect on protein function for the non-synonymous variants was determined with three bioinformatics servers. Three gene variants were identified in the HR gene of the evaluated patients. The benign polymorphism c.1010G > A p.(Gly337Asp) (rs12675375) had been previously reported, whereas the variants c.750G > A p.(Gln250Gln) and c.3215T > A (Val1072AGlu) have not been described in other world populations. Both non-synonymous variants proved to be significant (p ≤ 0.05). The variant c.3215T > A p.(Val1072Glu) is of particular interest due to its deleterious effect on the structure and function of the protein; therefore, it could be considered a risk factor for the development of AA. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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12 pages, 2139 KiB  
Communication
Proof-of-Concept Analysis of B Cell Receptor Repertoire in COVID-19 Patients Undergoing ECMO by Single-Cell V(D)J and Gene Expression Sequencing
by Alessia Gallo, Nicola Cuscino, Claudia Carcione, Rosalia Busà, Pier Giulio Conaldi and Matteo Bulati
Curr. Issues Mol. Biol. 2023, 45(2), 1471-1482; https://doi.org/10.3390/cimb45020095 - 09 Feb 2023
Viewed by 1808
Abstract
SARS-CoV-2, which causes COVID-19, has altered human activities all over the world and has become a global hazard to public health. Despite considerable advancements in pandemic containment techniques, in which vaccination played a key role, COVID-19 remains a global threat, particularly for frail [...] Read more.
SARS-CoV-2, which causes COVID-19, has altered human activities all over the world and has become a global hazard to public health. Despite considerable advancements in pandemic containment techniques, in which vaccination played a key role, COVID-19 remains a global threat, particularly for frail patients and unvaccinated individuals, who may be more susceptible to developing ARDS. Several studies reported that patients with COVID-19-related ARDS who were treated with ECMO had a similar survival rate to those with COVID-19-unrelated ARDS. In order to shed light on the potential mechanisms underlying the COVID-19 infection, we conducted this proof-of-concept study using single-cell V(D)J and gene expression sequencing of B cells to examine the dynamic changes in the transcriptomic BCR repertoire present in patients with COVID-19 at various stages. We compared a recovered and a deceased COVID-19 patient supported by ECMO with one COVID-19-recovered patient who did not receive ECMO treatment and one healthy subject who had never been infected previously. Our analysis revealed a downregulation of FXYD, HLA-DRB1, and RPS20 in memory B cells; MTATP8 and HLA-DQA1 in naïve cells; RPS4Y1 in activated B cells; and IGHV3-73 in plasma cells in COVID-19 patients. We further described an increased ratio of IgA + IgG to IgD + IgM, suggestive of an intensive memory antibody response, in the COVID ECMO D patient. Finally, we assessed a V(D)J rearrangement of heavy chain IgHV3, IGHJ4, and IGHD3/IGHD2 families in COVID-19 patients regardless of the severity of the disease. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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18 pages, 2859 KiB  
Article
Human Blood Extracellular Vesicles Activate Transcription of NF-kB-Dependent Genes in A549 Lung Adenocarcinoma Cells
by Yulya I. Savinovskaya, Anna A. Nushtaeva, Anna V. Savelyeva, Vitaliy V. Morozov, Elena I. Ryabchikova, Elena V. Kuligina, Vladimir A. Richter and Dmitriy V. Semenov
Curr. Issues Mol. Biol. 2022, 44(12), 6028-6045; https://doi.org/10.3390/cimb44120411 - 30 Nov 2022
Viewed by 1591
Abstract
Extracellular vesicles (EVs) produced by various cell types are heterogeneous in size and composition. Changes in the RNA sets of EVs in biological fluids are considered the basis for the development of new approaches to minimally invasive diagnostics and the therapy of human [...] Read more.
Extracellular vesicles (EVs) produced by various cell types are heterogeneous in size and composition. Changes in the RNA sets of EVs in biological fluids are considered the basis for the development of new approaches to minimally invasive diagnostics and the therapy of human diseases. In this study, EVs were obtained from the blood of healthy donors by centrifugation, followed by ultracentrifugation. It was shown that EVs consist of several populations including small exosome-like vesicles and larger microvesicle-like particles. The composition of EVs’ RNAs was determined. A549 lung adenocarcinoma cells were incubated with EV and the NGS analysis of differentially expressed genes was performed. During the incubation of A549 cells with EVs, the levels of mRNA encoding components for the NF-kB signaling pathway increased, as well as the expression of genes controlled by the NF-kB transcription factor. Overall, our results suggest that components of EVs trigger the NF-kB signaling cascade in A549 cells, leading to the transcription of genes including cytokines, adhesion molecules, cell cycle regulators, and cell survival factors. Our data provide insight into the interaction between blood EVs and human cells and can be used for designing new tools for the diagnosis and treatment of human diseases. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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7 pages, 2136 KiB  
Article
Usage of DNA Fingerprinting Technology to Check Sample Error and Contamination in Molecular Laboratories
by Dahui Qin, Mark Forster, Shital M. Gandhi, Ratilal Akabari, Zhong Zheng, Janella Lal and Katherine Lovinger
Curr. Issues Mol. Biol. 2022, 44(11), 5543-5549; https://doi.org/10.3390/cimb44110375 - 08 Nov 2022
Viewed by 2697
Abstract
Sample identification error is a severe medical error in clinical molecular diagnostic laboratories, which can lead to reporting the wrong results for the patient involved. Sample contamination can also lead to incorrect test reports. Avoiding sample identification error and sample contamination could be [...] Read more.
Sample identification error is a severe medical error in clinical molecular diagnostic laboratories, which can lead to reporting the wrong results for the patient involved. Sample contamination can also lead to incorrect test reports. Avoiding sample identification error and sample contamination could be life-saving. Sample switch and sample contamination could happen on laboratory bench works, especially when pipetting into multi-well plates. It is difficult to realize such errors during laboratory bench work. Laboratory staff may not be aware of such an error when it happens. DNA fingerprinting technology can be used to determine sample identity and subsequently identify sample switch and sample contamination in the laboratory. Our laboratory has explored the usage of this technology in our quality control process and successfully established that DNA fingerprinting can be used to monitor sample switch and sample contamination in next-generation sequencing and BCR/ABL1 real-time PCR bench work. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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8 pages, 1733 KiB  
Communication
Whole-Transcriptome Profiling on Small FFPE Samples: Which Sequencing Kit Should Be Used?
by Marc Hilmi, Lucile Armenoult, Mira Ayadi and Rémy Nicolle
Curr. Issues Mol. Biol. 2022, 44(5), 2186-2193; https://doi.org/10.3390/cimb44050148 - 13 May 2022
Viewed by 3371
Abstract
RNA sequencing (RNA-Seq) appears as a great tool with huge clinical potential, particularly in oncology. However, sufficient sample size is often a limiting factor and the vast majority of samples from patients with cancer are formalin-fixed paraffin-embedded (FFPE). To date, several sequencing kits [...] Read more.
RNA sequencing (RNA-Seq) appears as a great tool with huge clinical potential, particularly in oncology. However, sufficient sample size is often a limiting factor and the vast majority of samples from patients with cancer are formalin-fixed paraffin-embedded (FFPE). To date, several sequencing kits are proposed for FFPE samples yet no comparison on low quantities were performed. To select the most reliable, cost-effective, and relevant RNA-Seq approach, we applied five FFPE-compatible kits (based on 3′ capture, exome-capture and ribodepletion approaches) using 8 ng to 400 ng of FFPE-derived RNA and compared them to Nanostring on FFPE samples and to a reference PolyA (Truseq) approach on flash-frozen samples of the same tumors. We compared gene expression correlations and reproducibility. The Smarter Pico V3 ribodepletion approach appeared systematically the most comparable to Nanostring and Truseq (p < 0.001) and was a highly reproducible technique. In comparison with exome-capture and 3′ kits, the Smarter appeared more comparable to Truseq (p < 0.001). Overall, our results suggest that the Smarter is the most robust RNA-Seq technique to study small FFPE samples and 3′ Lexogen presents an interesting quality–price ratio for samples with less limiting quantities. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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12 pages, 1789 KiB  
Article
Comparison of Next-Generation Sequencing and Polymerase Chain Reaction for Personalized Treatment-Related Genomic Status in Patients with Metastatic Colorectal Cancer
by Wei-Chih Su, Yi-Chen Tsai, Hsiang-Lin Tsai, Tsung-Kun Chang, Tzu-Chieh Yin, Ching-Wen Huang, Yen-Cheng Chen, Ching-Chun Li, Po-Jung Chen, Yun-Ru Liu, Tsung-Han Hsieh and Jaw-Yuan Wang
Curr. Issues Mol. Biol. 2022, 44(4), 1552-1563; https://doi.org/10.3390/cimb44040106 - 05 Apr 2022
Cited by 3 | Viewed by 2449
Abstract
Personalized treatments based on the genetic profiles of tumors can simultaneously optimize efficacy and minimize toxicity, which is beneficial for improving patient outcomes. This study aimed to integrate gene alterations associated with predictive and prognostic outcomes in patients with metastatic colorectal cancer (mCRC) [...] Read more.
Personalized treatments based on the genetic profiles of tumors can simultaneously optimize efficacy and minimize toxicity, which is beneficial for improving patient outcomes. This study aimed to integrate gene alterations associated with predictive and prognostic outcomes in patients with metastatic colorectal cancer (mCRC) with polymerase chain reaction (PCR) and in-house next-generation sequencing (NGS) to detect KRAS, NRAS, and BRAF mutations. In the present study, 41 patients with mCRC were assessed between August 2017 and June 2019 at a single institution. The overall concordance between NGS and PCR results for detecting KRAS, NRAS, and BRAF mutations was considerably high (87.8–92.7%), with only 15 discrepant results between PCR and NGS. Our companion diagnostic test analyzes KRAS, NRAS, and BRAF as a panel of CRC molecular targets; therefore, it has the advantages of requiring fewer specimens and being more time and cost efficient than conventional testing for separate analyses, allowing for the simultaneous analysis of multiple genes. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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7 pages, 228 KiB  
Article
Next-Generation Sequencing of Connective Tissue Genes in Patients with Classical Ehlers-Danlos Syndrome
by Anna Junkiert-Czarnecka, Maria Pilarska-Deltow, Aneta Bąk, Marta Heise, Anna Latos-Bieleńska, Jacek Zaremba, Alicja Bartoszewska-Kubiak and Olga Haus
Curr. Issues Mol. Biol. 2022, 44(4), 1472-1478; https://doi.org/10.3390/cimb44040099 - 25 Mar 2022
Cited by 3 | Viewed by 3669
Abstract
Background: Ehlers-Danlos syndrome (EDS) is a common non-inflammatory, congenital connective tissue disorder. Classical type (cEDS) EDS is one of the more common forms, typically caused by mutations in the COL5A1 and COL5A2 genes, though causative mutations in the COL1A1 gene have also been [...] Read more.
Background: Ehlers-Danlos syndrome (EDS) is a common non-inflammatory, congenital connective tissue disorder. Classical type (cEDS) EDS is one of the more common forms, typically caused by mutations in the COL5A1 and COL5A2 genes, though causative mutations in the COL1A1 gene have also been described. Material and methods: The study group included 59 patients of Polish origin, diagnosed with cEDS. The analysis was performed on genomic DNA (gDNA) with NGS technology, using an Illumina sequencer. Thirty-five genes related to connective tissue were investigated. The pathogenicity of the detected variants was assessed by VarSome. Results: The NGS of 35 genes revealed variants within the COL5A1, COL5A2, COL1A1, and COL1A2 genes for 30 of the 59 patients investigated. Our panel detected no sequence variations for the remaining 29 patients. Discussion: Next-generation sequencing, with an appropriate multigene panel, showed great potential to assist in the diagnosis of EDS and other connective tissue disorders. Our data also show that not all causative genes giving rise to cEDS have been elucidated yet. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
17 pages, 648 KiB  
Article
Group Theory of Syntactical Freedom in DNA Transcription and Genome Decoding
by Michel Planat, Marcelo M. Amaral, Fang Fang, David Chester, Raymond Aschheim and Klee Irwin
Curr. Issues Mol. Biol. 2022, 44(4), 1417-1433; https://doi.org/10.3390/cimb44040095 - 22 Mar 2022
Cited by 3 | Viewed by 2448
Abstract
Transcription factors (TFs) are proteins that recognize specific DNA fragments in order to decode the genome and ensure its optimal functioning. TFs work at the local and global scales by specifying cell type, cell growth and death, cell migration, organization and timely tasks. [...] Read more.
Transcription factors (TFs) are proteins that recognize specific DNA fragments in order to decode the genome and ensure its optimal functioning. TFs work at the local and global scales by specifying cell type, cell growth and death, cell migration, organization and timely tasks. We investigate the structure of DNA-binding motifs with the theory of finitely generated groups. The DNA ‘word’ in the binding domain—the motif—may be seen as the generator of a finitely generated group Fdna on four letters, the bases A, T, G and C. It is shown that, most of the time, the DNA-binding motifs have subgroup structures close to free groups of rank three or less, a property that we call ‘syntactical freedom’. Such a property is associated with the aperiodicity of the motif when it is seen as a substitution sequence. Examples are provided for the major families of TFs, such as leucine zipper factors, zinc finger factors, homeo-domain factors, etc. We also discuss the exceptions to the existence of such DNA syntactical rules and their functional roles. This includes the TATA box in the promoter region of some genes, the single-nucleotide markers (SNP) and the motifs of some genes of ubiquitous roles in transcription and regulation. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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Review

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16 pages, 1455 KiB  
Review
A Bioinformatics Toolkit for Next-Generation Sequencing in Clinical Oncology
by Simon Cabello-Aguilar, Julie A. Vendrell and Jérôme Solassol
Curr. Issues Mol. Biol. 2023, 45(12), 9737-9752; https://doi.org/10.3390/cimb45120608 - 04 Dec 2023
Viewed by 1657
Abstract
Next-generation sequencing (NGS) has taken on major importance in clinical oncology practice. With the advent of targeted therapies capable of effectively targeting specific genomic alterations in cancer patients, the development of bioinformatics processes has become crucial. Thus, bioinformatics pipelines play an essential role [...] Read more.
Next-generation sequencing (NGS) has taken on major importance in clinical oncology practice. With the advent of targeted therapies capable of effectively targeting specific genomic alterations in cancer patients, the development of bioinformatics processes has become crucial. Thus, bioinformatics pipelines play an essential role not only in the detection and in identification of molecular alterations obtained from NGS data but also in the analysis and interpretation of variants, making it possible to transform raw sequencing data into meaningful and clinically useful information. In this review, we aim to examine the multiple steps of a bioinformatics pipeline as used in current clinical practice, and we also provide an updated list of the necessary bioinformatics tools. This resource is intended to assist researchers and clinicians in their genetic data analyses, improving the precision and efficiency of these processes in clinical research and patient care. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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8 pages, 864 KiB  
Case Report
Whole Genome Sequencing of a Chlamydia trachomatis Strain Responsible for a Case of Rectal Lymphogranuloma Venereum in Italy
by Antonella Marangoni, Stefano Amadesi, Marielle Ezekielle Djusse, Claudio Foschi, Valeria Gaspari, Tiziana Lazzarotto and Paolo Gaibani
Curr. Issues Mol. Biol. 2023, 45(3), 1852-1859; https://doi.org/10.3390/cimb45030119 - 23 Feb 2023
Cited by 1 | Viewed by 1529
Abstract
Lymphogranuloma venereum (LGV) is a systemic sexually transmitted infection caused by Chlamydia trachomatis serovars L1 to L3. The current LGV cases in Europe are mainly characterized by an anorectal syndrome, spreading within men who have sex with men (MSM). Whole-genome sequencing of LGV [...] Read more.
Lymphogranuloma venereum (LGV) is a systemic sexually transmitted infection caused by Chlamydia trachomatis serovars L1 to L3. The current LGV cases in Europe are mainly characterized by an anorectal syndrome, spreading within men who have sex with men (MSM). Whole-genome sequencing of LGV strains is crucial to the study of bacterial genomic variants and to improve strategies for contact tracing and prevention. In this study, we described the whole genome of a C. trachomatis strain (LGV/17) responsible for a case of rectal LGV. LGV/17 strain was isolated in 2017 in Bologna (North of Italy) from a HIV-positive MSM, presenting a symptomatic proctitis. After the propagation in LLC-MK2 cells, the strain underwent whole-genome sequencing by means of two platforms. Sequence type was determined using the tool MLST 2.0, whereas the genovariant was characterized by an ompA sequence evaluation. A phylogenetic tree was generated by comparing the LGV/17 sequence with a series of L2 genomes, downloaded from the NCBI website. LGV/17 belonged to sequence type ST44 and to the genovariant L2f. Nine ORFs encoding for polymorphic membrane proteins A-I and eight encoding for glycoproteins Pgp1-8 were detected in the chromosome and in the plasmid, respectively. LGV/17 was closely related to other L2f strains, even in the light of a not-negligible variability. The LGV/17 strain showed a genomic structure similar to reference sequences and was phylogenetically related to isolates from disparate parts of the world, indicative of the long-distance dynamics of transmission. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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9 pages, 2223 KiB  
Case Report
Identifying the Carcinogenic Mechanism of Malignant Struma Ovarii Using Whole-Exome Sequencing and DNA Methylation Analysis
by Hitomi Yamashita, Kentaro Nakayama, Kosuke Kanno, Tomoka Ishibashi, Masako Ishikawa, Seiya Sato, Koji Iida, Sultana Razia and Satoru Kyo
Curr. Issues Mol. Biol. 2023, 45(3), 1843-1851; https://doi.org/10.3390/cimb45030118 - 23 Feb 2023
Cited by 1 | Viewed by 1515
Abstract
Background: Since malignant struma ovarii is a very rare disease, its carcinogenic mechanism has not been elucidated. Here, we sought to identify the genetic lesions that may have led to the carcinogenesis of a rare case of malignant struma ovarii (follicular carcinoma) with [...] Read more.
Background: Since malignant struma ovarii is a very rare disease, its carcinogenic mechanism has not been elucidated. Here, we sought to identify the genetic lesions that may have led to the carcinogenesis of a rare case of malignant struma ovarii (follicular carcinoma) with peritoneal dissemination. Methods: DNA was extracted from the paraffin-embedded sections of normal uterine tissues and malignant struma ovarii for genetic analysis. Whole-exome sequencing and DNA methylation analysis were then performed. Results: Germline variants of RECQL4, CNTNAP2, and PRDM2, which are tumor-suppressor genes, were detected by whole-exome sequencing. Somatic uniparental disomy (UPD) was also observed in these three genes. Additionally, the methylation of FRMD6-AS2, SESN3, CYTL1, MIR4429, HIF3A, and ATP1B2, which are associated with tumor growth suppression, was detected by DNA methylation analysis. Conclusions: Somatic UPD and DNA methylation in tumor suppressor genes may be associated with the pathogenesis of malignant struma ovarii. To our knowledge, this is the first report of whole-exome sequencing and DNA methylation analysis in malignant struma ovarii. Genetic and DNA methylation analysis may help elucidate the mechanism of carcinogenesis in rare diseases and guide treatment decisions. Full article
(This article belongs to the Special Issue Next-Generation Sequencing (NGS) Technique and Personalized Medicine)
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