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Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Informatics".

Deadline for manuscript submissions: closed (30 September 2024) | Viewed by 7374

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Special Issue Editor

Dr. Behzad Imanian

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Guest Editor

1. Institute for Food Safety and Health, Illinois Institute of Technology, Bedford Park, IL 60501, USA
2. Food Science and Nutrition Department, Illinois Institute of Technology, Chicago, IL 60616, USA
Interests: gene and genome evolution; gene transfers (HGT & EGT); organelle genome, transcriptome, proteome; organelle metabolism; evolution of biochemical pathways; endosymbiosis; reductive evolution; evolutionary history of eukaryotes; evolution of pathogenicity; food safety and microorganisms

Special Issue Information

Dear Colleagues,

Since its emergence approximately two decades ago, High-Throughput or Next-Generation Sequencing (HTS/NGS) has continuously advanced, significantly influencing a wide array of scientific disciplines, including biology, genetics, genomics, physiology, ecology, chemistry, and biochemistry. Researchers and practitioners worldwide, working in governmental, academic, and industrial sectors, have adopted this technology as an essential tool for unraveling profound scientific inquiries, as well as in response to the serious challenges of our times that have tremendous implications for human health and the well-being of plants, animals, and even our planet.

HTS is a fast-moving technology characterized by ongoing improvements in quality, length, depth, and the number of sequences, complemented by innovative breakthroughs in biochemistry, design, and engineering. These developments have led to the introduction of more powerful instruments with increasing throughput power year by year. The numerous applications of HTS, such as Whole-Genome Sequencing (WGS), RNA-Seq, and Amplicon or Shotgun Mass Sequencing, have greatly contributed to the advancement of metagenomics, transcriptomics, proteomics, metabolomics, and phylogenomics, fostering the rapid development of novel investigative, diagnostic, and analytical methods, thus harnessing the full potential of this transformative technology.

Finally, HTS has become one of the main contributors to ‘big data’, a valuable resource for predictive modeling through the application of machine learning and artificial intelligence (AI). In this Special Issue, we cordially invite you to share your HTS-based research and review papers, emphasizing your latest discoveries and the transformative power and influence of this technology, as well as its numerous applications within varied domains of study and organizations.

Dr. Behzad Imanian
Guest Editor

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Keywords

high-throughput sequencing
whole-genome sequencing
big data
machine learning
artificial intelligence
predictive modeling
transformative technology
investigative and diagnostic tools

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Published Papers (7 papers)

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Research

15 pages, 2232 KiB

Open AccessArticle

Molecular Composition and Kinetics of B Cells During Ibrutinib Treatment in Patients with Chronic Lymphocytic Leukemia

by Sólja Remisdóttir Veyhe, Oriane Cédile, Sara Kamuk Dahlmann, Jakub Krejcik, Niels Abildgaard, Thor Høyer, Michael Boe Møller, Mads Thomassen, Karen Juul-Jensen, Henrik Frederiksen, Karen Dybkær, Marcus Høy Hansen and Charlotte Guldborg Nyvold

Int. J. Mol. Sci. 2024, 25(23), 12569; https://doi.org/10.3390/ijms252312569 - 22 Nov 2024

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of B cells due to constitutive B-cell receptor (BCR) signaling, leading to apoptosis resistance and increased proliferation. This study evaluates the effects of the Bruton Tyrosine Kinase (BTK) inhibitor ibrutinib on the molecular composition, clonality, and kinetics of B cells during treatment in CLL patients. Employing a multi-omics approach of up to 3.2 years of follow-up, we analyzed data from 24 CLL patients, specifically focusing on nine patients treated with ibrutinib monotherapy. In this study, clonal stability was observed within the ibrutinib-treated group following an effective initial clinical response, where clonotype frequencies of residual CLL cells remained high and stable, ranging from 74.9% at 1.5 years to 87.7% at approximately 3 years. In contrast, patients treated with the B-cell lymphoma 2 (BCL2) inhibitor venetoclax exhibited substantial reductions in clonal frequencies, approaching molecular eradication. Deep whole-exome sequencing revealed minimal genomic progression in the ibrutinib group, maintaining somatic drivers and variant allele frequencies (VAF) above 0.2 throughout treatment. At the single-cell level, the NF-κB pathway inhibition and apoptotic signals were detected or even augmented during treatment in ibrutinib-treated patients. These findings may corroborate the role of ibrutinib in stabilizing the genomic landscape of CLL cells, preventing significant genomic evolution despite maintaining a high clonal burden within the residual B-cell compartment. Full article

(This article belongs to the Special Issue Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research)

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18 pages, 2703 KiB

Open AccessArticle

Single Laboratory Evaluation of the Q20+ Nanopore Sequencing Kit for Bacterial Outbreak Investigations

by Maria Hoffmann, Jay Hee Jang, Sandra M. Tallent and Narjol Gonzalez-Escalona

Int. J. Mol. Sci. 2024, 25(22), 11877; https://doi.org/10.3390/ijms252211877 - 5 Nov 2024

Viewed by 502

Abstract

Leafy greens are a significant source of produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States, with agricultural water often implicated as a potential source. Current FDA outbreak detection protocols are time-consuming and rely on sequencing methods performed in costly equipment. This study evaluated the potential of Oxford Nanopore Technologies (ONT) with Q20+ chemistry as a cost-effective, rapid, and accurate method for identifying and clustering foodborne pathogens. The study focuses on assessing whether ONT Q20+ technology could facilitate near real-time pathogen identification, including SNP differences, serotypes, and antimicrobial resistance genes. This pilot study evaluated different combinations of two DNA extraction methods (Maxwell RSC Cultured Cell DNA kit and Monarch high molecular weight extraction kits) and two ONT library preparation protocols (ligation and the rapid barcoding sequencing kit) using five well-characterized strains representing diverse foodborne pathogens. High-quality, closed bacterial genomes were obtained from all combinations of extraction and sequencing kits. However, variations in assembly length and genome completeness were observed, indicating the need for further optimization. In silico analyses demonstrated that Q20+ nanopore sequencing chemistry accurately identified species, genotype, and virulence factors, with comparable results to Illumina sequencing. Phylogenomic clustering showed that ONT assemblies clustered with reference genomes, though some indels and SNP differences were observed, likely due to sequencing and analysis methodologies rather than inherent genetic variation. Additionally, the study evaluated the impact of a change in the sampling rates from 4 kHz (260 bases pair second) to 5 kHz (400 bases pair second), finding no significant difference in sequencing accuracy. This evaluation workflow offers a framework for evaluating novel technologies for use in surveillance and foodborne outbreak investigations. Overall, the evaluation demonstrated the potential of ONT Q20+ nanopore sequencing chemistry to assist in identifying the correct strain during outbreak investigations. However, further research, validation studies, and optimization efforts are needed to address the observed limitations and fully realize the technology’s potential for improving public health outcomes and enabling more efficient responses to foodborne disease threats. Full article

(This article belongs to the Special Issue Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research)

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12 pages, 1275 KiB

Open AccessArticle

The Next Frontier in Tuberculosis Investigation: Automated Whole Genome Sequencing for Mycobacterium tuberculosis Analysis

by Justin H. J. Ng, Lina Castro, Andrew Gorzalski, Adam Allred, Danielle Siao, Edwina Wong, Andrew Lin, Shadi Shokralla, Mark Pandori, Godfred Masinde and Ramin Khaksar

Int. J. Mol. Sci. 2024, 25(14), 7909; https://doi.org/10.3390/ijms25147909 - 19 Jul 2024

Viewed by 1296

Abstract

A fully automated bacteria whole genome sequencing (WGS) assay was evaluated to characterize Mycobacterium tuberculosis (MTB) and non-tuberculosis Mycobacterium (NTM) clinical isolates. The results generated were highly reproducible, with 100% concordance in species and sub-lineage classification and 92% concordance between antimicrobial resistance (AMR) genotypic and phenotypic profiles. Using extracted deoxyribonucleic acid (DNA) from MTB clinical isolates as starting material, these findings demonstrate that a fully automated WGS assay, with a short turnaround time of 24.5 hours, provides timely and valuable insights into MTB outbreak investigation while providing reliable genotypic AMR profiling consistent with traditional antimicrobial susceptibility tests (AST). This study establishes a favorable proposition for the adoption of end-to-end fully automated WGS solutions for decentralized MTB diagnostics, thereby aiding in World Health Organization’s (WHO) vision of tuberculosis eradication. Full article

(This article belongs to the Special Issue Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research)

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16 pages, 8280 KiB

Open AccessArticle

Genetic Diversity and Population Structure of Rhodeus uyekii in the Republic of Korea Revealed by Microsatellite Markers from Whole Genome Assembly

by Kang-Rae Kim, So Young Park, Ju Hui Jeong, Yujin Hwang, Heesoo Kim, Mu-Sung Sung and Jeong-Nam Yu

Int. J. Mol. Sci. 2024, 25(12), 6689; https://doi.org/10.3390/ijms25126689 - 18 Jun 2024

Viewed by 847

Abstract

This study is the first report to characterize the Rhodus uyekii genome and study the development of microsatellite markers and their markers applied to the genetic structure of the wild population. Genome assembly was based on PacBio HiFi and Illumina HiSeq paired-end sequencing, resulting in a draft genome assembly of R. uyekii. The draft genome was assembled into 2652 contigs. The integrity assessment of the assemblies indicates that the quality of the draft assemblies is high, with 3259 complete BUSCOs (97.2%) in the database of Verbrata. A total of 31,166 predicted protein-coding genes were annotated in the protein database. The phylogenetic tree showed that R. uyekii is a close but distinct relative of Onychostoma macrolepis. Among the 10 fish genomes, there were significant gene family expansions (8–2387) and contractions (16–2886). The average number of alleles amplified by the 21 polymorphic markers ranged from 6 to 23, and the average PIC value was 0.753, which will be useful for evolutionary and genetic analysis. Using population genetic analysis, we analyzed genetic diversity and the genetic structures of 120 individuals from 6 populations. The average number of alleles per population ranged from 7.6 to 9.9, observed heterozygosity ranged from 0.496 to 0.642, and expected heterozygosity ranged from 0.587 to 0.783. Discriminant analysis of principal components According to the analysis method, the population was divided into three populations (BS vs. DC vs. GG, GC, MS, DC). In conclusion, our study provides a useful resource for comparative genomics, phylogeny, and future population studies of R. uyekii. Full article

(This article belongs to the Special Issue Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research)

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14 pages, 267 KiB

Open AccessArticle

Evaluation of Whole Genome Sequencing-Based Predictions of Antimicrobial Resistance to TB First Line Agents: A Lesson from 5 Years of Data

by Meenu Kaushal Sharma, Michael Stobart, Pierre-Marie Akochy, Heather Adam, Debra Janella, Melissa Rabb, Mohey Alawa, Inna Sekirov, Gregory J. Tyrrell and Hafid Soualhine

Int. J. Mol. Sci. 2024, 25(11), 6245; https://doi.org/10.3390/ijms25116245 - 6 Jun 2024

Cited by 1 | Viewed by 1065

Abstract

Phenotypic susceptibility testing of the Mycobacterium tuberculosis complex (MTBC) isolate requires culture growth, which can delay rapid detection of resistant cases. Whole genome sequencing (WGS) and data analysis pipelines can assist in predicting resistance to antimicrobials used in the treatment of tuberculosis (TB). This study compared phenotypic susceptibility testing results and WGS-based predictions of antimicrobial resistance (AMR) to four first-line antimicrobials—isoniazid, rifampin, ethambutol, and pyrazinamide—for MTBC isolates tested between the years 2018–2022. For this 5-year retrospective analysis, the WGS sensitivity for predicting resistance for isoniazid, rifampin, ethambutol, and pyrazinamide using Mykrobe was 86.7%, 100.0%, 100.0%, and 47.8%, respectively, and the specificity was 99.4%, 99.5%, 98.7%, and 99.9%, respectively. The predictive values improved slightly using Mykrobe corrections applied using TB Profiler, i.e., the WGS sensitivity for isoniazid, rifampin, ethambutol, and pyrazinamide was 92.31%, 100%, 100%, and 57.78%, respectively, and the specificity was 99.63%. 99.45%, 98.93%, and 99.93%, respectively. The utilization of WGS-based testing addresses concerns regarding test turnaround time and enables analysis for MTBC member identification, antimicrobial resistance prediction, detection of mixed cultures, and strain genotyping, all through a single laboratory test. WGS enables rapid resistance detection compared to traditional phenotypic susceptibility testing methods using the WHO TB mutation catalog, providing an insight into lesser-known mutations, which should be added to prediction databases as high-confidence mutations are recognized. The WGS-based methods can support TB elimination efforts in Canada and globally by ensuring the early start of appropriate treatment, rapidly limiting the spread of TB outbreaks. Full article

(This article belongs to the Special Issue Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research)

14 pages, 3757 KiB

Open AccessArticle

Microbial Detoxification of Sediments Underpins Persistence of Zostera marina Meadows

by Yuki Nakashima, Takumi Sonobe, Masashi Hanada, Goushi Kitano, Yoshimitsu Sonoyama, Katsumi Iwai, Takashi Kimura and Masataka Kusube

Int. J. Mol. Sci. 2024, 25(10), 5442; https://doi.org/10.3390/ijms25105442 - 16 May 2024

Cited by 1 | Viewed by 1074

Abstract

Eelgrass meadows have attracted much attention not only for their ability to maintain marine ecosystems as feeding grounds for marine organisms but also for their potential to store atmospheric and dissolved CO₂ as blue carbon. This study comprehensively evaluated the bacterial and chemical data obtained from eelgrass sediments of different scales along the Japanese coast to investigate the effect on the acclimatization of eelgrass. Regardless of the eelgrass habitat, approximately 1% Anaerolineales, Babeliales, Cytophagales, and Phycisphaerales was present in the bottom sediment. Sulfate-reducing bacteria (SRB) were present at 3.69% in eelgrass sediment compared to 1.70% in bare sediment. Sulfur-oxidizing bacteria (SOB) were present at 2.81% and 1.10% in the eelgrass and bare sediment, respectively. Bacterial composition analysis and linear discriminant analysis revealed that SOB detoxified H₂S in the eelgrass meadows and that the larger-scale eelgrass meadows had a higher diversity of SOB. Our result indicated that there were regional differences in the system that detoxifies H₂S in eelgrass meadows, either microbial oxidation mediated by SOB or O₂ permeation via the physical diffusion of benthos. However, since bacterial flora and phylogenetic analyses cannot show bias and/or causality due to PCR, future kinetic studies on microbial metabolism are expected. Full article

(This article belongs to the Special Issue Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research)

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15 pages, 602 KiB

Open AccessArticle

Comparative Analyses of Targeted Myeloid Cancer Next-Generation Sequencing Panel in Fresh Blood, Bone Marrow and FFPE Material

by Andrea Daniela Hobeck, Sophia Wendt, Saskia Krohn, Gudrun Knuebel, Stephan Bartels, Elisa Schipper, Christian Junghanss and Hugo Murua Escobar

Int. J. Mol. Sci. 2024, 25(6), 3534; https://doi.org/10.3390/ijms25063534 - 21 Mar 2024

Viewed by 1317

Abstract

Next-generation sequencing is a vital tool for personalized diagnostics and therapies in cancer. Despite numerous advantages, the method depends on multiple parameters regarding the sample material, e.g., sample fixation. A panel’s ability to ensure balanced pre-amplification of the regions of interest is challenging, especially in targeted sequencing approaches, but of significant importance to its applicability across hematological malignancies and solid tumors. This study comparatively evaluated the technical performance of the commercially available Oncomine^TM Myeloid Panel in fresh and Formalin-fixed paraffin-embedded (FFPE) material by using an Ion Torrent™ Personal Genome Machine™ System and Ion GeneStudio S5 System platform. In total, 114 samples were analyzed, including 55 fresh materials and 59 FFPE samples. Samples were sequenced with a minimum of one million reads. Amplicons with coverage below 400 reads were classified as underperforming. In fresh material, 49/526 amplicons were identified as performing insufficiently, corresponding with 18 genes. Using FFPE material, 103/526 amplicons underperformed. Independent of input material, regions in 27 genes, including ASXL1, BCOR and BRAF, did not match quality parameters. Subsequently, exemplary mutations were extracted from the Catalogue of Somatic Mutations in Cancer database. This technical evaluation of the Oncomine^TM Myeloid Panel identified amplicons that do not achieve adequate coverage levels and which need to be considered when interpreting sequencing. Full article

(This article belongs to the Special Issue Next-Generation Sequencing and Whole-Genome Sequencing in Molecular Research)

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