Third-generation Sequencing Technologies Research Articles

Comprehensive transcriptome sequencing of silkworm Midguts: Uncovering extensive isoform diversity and alternative splicing in BmNPV-Sensitive and BmNPV-resistant strains

The silkworm, Bombyx mori, stands out as one of the few economically valuable insects within the realm of model organisms. However, Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat, decreasing the quality and quantity of silkworm cocoons. Over the past few decades, a multitude of researchers has delved into the mechanisms that underlie silkworm resistance to BmNPV, employing diverse methodologies and approaching the problem from various angles. Despite this extensive research, the role of alternative splicing (AS) in the silkworm's response to BmNPV infection has been largely unexplored. This study leveraged both third-generation (Oxford Nanopore Technologies) and second-generation (Illumina) high-throughput sequencing technologies to meticulously identify and analyze AS patterns in the context of BmNPV response, utilizing two distinct silkworm strains—the susceptible strain 306 and the resistant strain NB. Consequently, we identified five crucial genes (Dsclp, LOC692903, LOC101743583, LOC101742498, LOC101743809) that are linked to the response to BmNPV infection through AS and differential expression. Additionally, a thorough comparative analysis was conducted on their diverse transcriptomic expression profiles, including alternative polyadenylation, simple sequence repeats, and transcription factors.

DNA Barcoding for Species Identification of Moss-Dwelling Invertebrates: Performance of Nanopore Sequencing and Coverage in Reference Database

In view of the current biodiversity crisis and our need to preserve and improve ecosystem functioning, efficient means for characterizing and monitoring biodiversity are required. DNA barcoding, especially when coupled with new sequencing technologies, is a promising method that can, in principle, also be employed by taxonomic lay people. In this study we compare the performance of DNA barcoding by means of a third-generation sequencing technology, nanopore sequencing with classical Sanger sequencing, based on a sample of invertebrates collected from moss pads in a bog in Austria. We find that our nanopore sequencing pipeline generates DNA barcodes that are at least as good as barcodes generated with Sanger sequencing, with the MinION producing better results than the Flongle flowcell. We further find that while many arthropod taxa are well covered in the international reference DNA barcode database BOLD, this clearly is not the case for important taxa like mites and springtails, which hampers large-scale biodiversity assessments. Based on examples from our study we further highlight which factors might be responsible for ambiguous species identification based on BOLD and how this can, at least partly, be solved.

SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms.

SQANTI3 is a tool designed for the quality control, curation and annotation of long-read transcript models obtained with third-generation sequencing technologies. Leveraging its annotation framework, SQANTI3 calculates quality descriptors of transcript models, junctions and transcript ends. With this information, potential artifacts can be identified and replaced with reliable sequences. Furthermore, the integrated functional annotation feature enables subsequent functional iso-transcriptomics analyses.

Gene sequencing technologies and their application in cancer research

The continuous development of gene sequencing technology has made the next-generation sequencing technology and the third-generation sequencing technology gradually applied to scientific research, which has greatly improved the efficiency and accuracy of sequencing and reduced the cost. The incidence of tumors in the human population continues to rise, making tumors gradually become one of the most serious diseases that endanger health. In view of the close relationship between gene mutations and tumorigenesis, the progress of gene sequencing technology has a profound impact on clinical research on tumors. In this thesis, literature analysis was used to introduce several gene sequencing technologies and compare them from multiple perspectives. In addition, the causes of tumorigenesis and the role of sequencing technology in the clinical research of human tumors were investigated. This paper found that the third-generation sequencing technology was more efficient than the next-generation sequencing technology, but it was difficult to be widely used due to its lower accuracy. Sequencing technology is of great help to tumor clinical research.

MCSS: microbial community simulator based on structure.

De novo assembly plays a pivotal role in metagenomic analysis, and the incorporation of third-generation sequencing technology can significantly improve the integrity and accuracy of assembly results. Recently, with advancements in sequencing technology (Hi-Fi, ultra-long), several long-read-based bioinformatic tools have been developed. However, the validation of the performance and reliability of these tools is a crucial concern. To address this gap, we present MCSS (microbial community simulator based on structure), which has the capability to generate simulated microbial community and sequencing datasets based on the structure attributes of real microbiome communities. The evaluation results indicate that it can generate simulated communities that exhibit both diversity and similarity to actual community structures. Additionally, MCSS generates synthetic PacBio Hi-Fi and Oxford Nanopore Technologies (ONT) long reads for the species within the simulated community. This innovative tool provides a valuable resource for benchmarking and refining metagenomic analysis methods. Code available at: https://github.com/panlab-bio/mcss.

Advancements in HLA Typing Techniques and their Impact on Transplantation Medicine.

HLA typing serves as a standard practice in hematopoietic stem cell transplantation to ensure compatibility between donors and recipients, preventing the occurrence of allograft rejection and graft-versus-host disease. Conventional laboratory methods that have been widely employed in the past few years, including Sequence-Specific Primer PCR and Sequencing Based Typing (SBT), currently face the risk of becoming obsolete. This risk stems not only from the extensive diversity within HLA genes but also from the rapid advancement of Next Generation Sequencing and third-generation sequencing technologies. Third-generation sequencing systems like Single Molecule Real Time (SMRT) sequencing and Oxford Nanopore (ONT) sequencing have the capability to analyze long-read sequences that span entire intronic-exonic regions of HLA genes, effectively addressing challenges related to HLA ambiguity and the phasing of multiple short-read fragments. The growing dominance of these advanced sequencers in HLA typing is expected to solidify further through ongoing refinements, cost reduction, and error rate minimization. This review focuses on hematopoietic stem cell transplantation (HSCT) and explores prospective advancements and application of HLA DNA typing techniques. It explores how the adoption of third-generation sequencing technologies can revolutionize the field by offering improved accuracy, reduced ambiguity, and enhanced assessment of compatibility in HSCT. Embracing these cutting-edge technologies is essential to advancing the success rates and outcomes of hematopoietic stem cell transplantation. This review underscores the importance of staying at the forefront of HLA typing techniques to ensure the best possible outcomes for patients undergoing HSCT.

When Livestock Genomes Meet Third-Generation Sequencing Technology: From Opportunities to Applications.

Third-generation sequencing technology has found widespread application in the genomic, transcriptomic, and epigenetic research of both human and livestock genetics. This technology offers significant advantages in the sequencing of complex genomic regions, the identification of intricate structural variations, and the production of high-quality genomes. Its attributes, including long sequencing reads, obviation of PCR amplification, and direct determination of DNA/RNA, contribute to its efficacy. This review presents a comprehensive overview of third-generation sequencing technologies, exemplified by single-molecule real-time sequencing (SMRT) and Oxford Nanopore Technology (ONT). Emphasizing the research advancements in livestock genomics, the review delves into genome assembly, structural variation detection, transcriptome sequencing, and epigenetic investigations enabled by third-generation sequencing. A comprehensive analysis is conducted on the application and potential challenges of third-generation sequencing technology for genome detection in livestock. Beyond providing valuable insights into genome structure analysis and the identification of rare genes in livestock, the review ventures into an exploration of the genetic mechanisms underpinning exemplary traits. This review not only contributes to our understanding of the genomic landscape in livestock but also provides fresh perspectives for the advancement of research in this domain.

A gene cluster encoding a nonribosomal peptide synthetase-like enzyme catalyzes γ-aromatic butenolides

Sea cucumber-derived fungi have attracted much attention due to their capacity to produce an incredible variety of secondary metabolites. Genome-wide information on Aspergillus micronesiensis H39 obtained using third-generation sequencing technology (PacBio-SMRT) showed that the strain contains nonribosomal peptide synthetase (NRPS)-like gene clusters, which aroused our interest in mining its secondary metabolites. 11 known compounds (1–11), including two γ-aromatic butenolides (γ-AB) and five cytochalasans, were isolated from A. micronesiensis H39. The structures of the compounds were determined by NMR and ESIMS, and comparison with those reported in the literature. From the perspective of biogenetic origins, the γ-butyrolactone core of compounds 1 and 2 was assembled by NRPS-like enzyme. All of the obtained compounds showed no inhibitory activity against drug-resistant bacteria and fungi, as well as compounds 1 and 2 had no anti-angiogenic activity against zebrafish.

Innovations in Molecular Biology-Cutting-Edge Breakthroughs in Molecular Genetics

The field of molecular biology has experienced significant breakthroughs in recent years, driven by cutting-edge technologies and innovative research strategies. This abstract provides a concise overview of some key advancement that has shaped the landscape of molecular biology. One prominent area of progress involves the CRISPR-Cas9 gene editing system, which has revolutionized genetic manipulation. Researchers have refined and expanded its applications, enabling precise modifications to the genome for therapeutic purposes, functional genomics, and the development of genetically modified organisms. In the realm of nucleic acid sequencing, the advent of third-generation sequencing technologies has enhanced the accuracy and efficiency of deciphering complex genomes. Single-cell sequencing techniques have provided unprecedented insights into cellular heterogeneity, unraveling diverse cell populations within tissues and shedding light on the intricacies of developmental processes and disease progression. The integration of omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, has propelled systems biology to new heights. This holistic approach allows for a comprehensive understanding of biological systems, unveiling intricate molecular networks and signaling pathways. Advanced computational methods and artificial intelligence applications have played a pivotal role in processing and interpreting the vast amounts of data generated by these high-throughput techniques. Furthermore, the exploration of the microbiome's role in health and disease has gained momentum. Advances in metagenomics have enabled a deeper understanding of microbial communities, their interactions, and their impact on host physiology. The identification of specific microbial signatures associated with various diseases has opened avenues for novel therapeutic interventions and personalized medicine. Conclusion: Recent advances in molecular biology have transformed the field, offering unprecedented opportunities for scientific discovery and medical applications. The integration of cutting-edge technologies and interdisciplinary approaches continues to propel molecular biology forward, paving the way for new insights into the complexities of life at the molecular level

Driving forces shaping the microbial ecology in meat packing plants.

Meat production is a complex system, continually receiving animals, water, air, and workers, all of which serve as carriers of bacteria. Selective pressures involved in different meat processing stages such as antimicrobial interventions and low temperatures, may promote the accumulation of certain residential microbiota in meat cutting facilities. Bacteria including human pathogens from all these sources can contaminate meat surfaces. While significant advancements have been made in enhancing hygienic standards and pathogen control measures in meat plants, resulting in a notable reduction in STEC recalls and clinical cases, STEC still stands as a predominant contributor to foodborne illnesses associated with beef and occasionally with pork. The second-and third-generation sequencing technology has become popular in microbiota related studies and provided a better image of the microbial community in the meat processing environments. In this article, we reviewed the potential factors influencing the microbial ecology in commercial meat processing facilities and conducted a meta-analysis on the microbiota data published in the last 10 years. In addition, the mechanisms by which bacteria persist in meat production environments have been discussed with a focus on the significant human pathogen E. coli O157:H7 and generic E. coli, an indicator often used for the hygienic condition in food production.

Comparative analysis of the intestinal microbiota of black-necked cranes (Grus nigricollis) in different wintering areas.

Fecal microbiota is essential for host health because it increases digestive effectiveness. The crane species Grus nigricollis (G. nigricollis) is considered to be near threatened. The fecal microbial composition of crane is less understood, particularly in the Tibet, China. This study was performed to investigate the differences in fecal microbial composition and diversity of crane in different wintering areas using third-generation single-molecule real-time sequencing technology in the Tibet, China. According to the findings, 20 samples were used to generate 936 bacterial amplicon sequence variants (ASVs) and 1,800 fungal ASVs, only 4 bacterial ASVs and 20 fungal ASVs were shared in four distinct locations. Firmicutes were the dominant bacterial phylum in all samples, and Ascomycota and Basidiomycota were the dominant fungal phylum. At the genus level, Lactobacillus was the dominant genus in Linzhi City (LZ), Shannan City (SN), and Lasa City (LS), whereas Megamonas was the dominant genus in Rikaze City (RKZ). Naganishia and Mycosphaerella were the dominant fungal genera in SN and RKZ. Mycosphaerella and Tausonia were the dominant fungal genera in LZ. Naganishia and Fusarium were the dominant fungal genera in LS. And the fecal microbial composition varied between the four groups, as shown by the underweighted pair-group method with arithmetic means and principal coordinates analysis. This study offers a theoretical basis for understanding the fecal microbial composition of crane.

Third-generation sequencing identified two rare α-chain variants leading to hemoglobin variants in Chinese population.

Rare and novel variants of HBA1/2 and HBB genes resulting in thalassemia and hemoglobin (Hb) variants have been increasingly identified. Our goal was to identify two rare Hb variants in Chinese population using third-generation sequencing (TGS) technology. Enrolled in this study were two Chinese families from Fujian Province. Hematological screening was conducted using routine blood analysis and Hb capillary electrophoresis analysis. Routine thalassemia gene testing was carried out to detect the common mutations of α- and β-thalassemia in Chinese population. Rare or novel α- and β-globin gene variants were further investigated by TGS. The proband of family 1 was a female aged 32, with decreased levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), Hb A2, and abnormal Hb bands in zone 5 and zone 12. No common thalassemia mutations were detected by routine thalassemia analysis, while a rare α-globin gene variant Hb Jilin [α139(HC1)Lys>Gln (AAA>CAA); HBA2:c.418A>C] was identified by TGS. Subsequent pedigree analysis showed that the proband's son also harbored the Hb Jilin variant with slightly low levels of MCH, Hb A2, and abnormal Hb bands. The proband of family 2 was a male at 41 years of age, exhibiting normal MCV and MCH, but a low level of Hb A2 and an abnormal Hb band in zone 12 without any common α- and β-thalassemia mutations. The subsequent TGS detection demonstrated a rare Hb Beijing [α16(A14)Lys>Asn (AAG>AAT); HBA2:c.51G>T] variant in HBA2 gene. In this study, for the first time, we present two rare Hb variants of Hb Jilin and Hb Beijing in Fujian Province, Southeast China, using TGS technology.

Direct Determination of the Structure of Single Biopolymer Molecules Using Nanopore Sequencing.

This review highlights operational principles, features, and modern aspects of the development of third-generation sequencing technology of biopolymers focusing on the nucleic acids analysis, namely the nanopore sequencing system. Basics of the method and technical solutions used for its realization are considered, from the first works showing the possibility of creation of these systems to the easy-to-handle procedure developed by Oxford Nanopore Technologies company. Moreover, this review focuses on applications, which were developed and realized using equipment developed by the Oxford Nanopore Technologies, including assembly of whole genomes, methagenomics, direct analysis of the presence of modified bases.

Viral oncogenes, viruses, and cancer: a third-generation sequencing perspective on viral integration into the human genome.

The link between viruses and cancer has intrigued scientists for decades. Certain viruses have been shown to be vital in the development of various cancers by integrating viral DNA into the host genome and activating viral oncogenes. These viruses include the Human Papillomavirus (HPV), Hepatitis B and C Viruses (HBV and HCV), Epstein-Barr Virus (EBV), and Human T-Cell Leukemia Virus (HTLV-1), which are all linked to the development of a myriad of human cancers. Third-generation sequencing technologies have revolutionized our ability to study viral integration events at unprecedented resolution in recent years. They offer long sequencing capabilities along with the ability to map viral integration sites, assess host gene expression, and track clonal evolution in cancer cells. Recently, researchers have been exploring the application of Oxford Nanopore Technologies (ONT) nanopore sequencing and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) sequencing in cancer research. As viral integration is crucial to the development of cancer via viruses, third-generation sequencing would provide a novel approach to studying the relationship interlinking viral oncogenes, viruses, and cancer. This review article explores the molecular mechanisms underlying viral oncogenesis, the role of viruses in cancer development, and the impact of third-generation sequencing on our understanding of viral integration into the human genome.

Characterizing viral species in mosquitoes (Culicidae) in the Colombian Orinoco: insights from a preliminary metagenomic study

Mosquitoes (Diptera: Culicidae) are primary vectors of arthropod-borne viruses (arboviruses) that pose significant public health threats. Recent advances in sequencing technology emphasize the importance of understanding the arboviruses and insect-specific viruses (ISVs) hosted by mosquitoes, collectively called the “virome”. Colombia, a tropical country with favorable conditions for the development and adaptation of multiple species of Culicidae, offers a favorable scenario for the transmission of epidemiologically important arboviruses. However, entomovirological surveillance studies are scarce in rural areas of the country, where humans, mosquitoes, and animals (both domestic and wild) coexist, leading to a higher risk of transmission of zoonotic diseases to humans. Thus, our study aimed to perform a preliminary metagenomic analysis of the mosquitoes of special relevance to public health belonging to the genera Ochlerotatus, Culex, Limatus, Mansonia, Psorophora, and Sabethes, within a rural savanna ecosystem in the Colombian Orinoco. We employed third-generation sequencing technology (Oxford Nanopore Technologies; ONT) to describe the virome of mosquitoes samples. Our results revealed that the virome was primarily shaped by insect-specific viruses (ISVs), with the Iflaviridae family being the most prevalent across all mosquito samples. Furthermore, we identified a group of ISVs that were common in all mosquito species tested, displaying the highest relative abundance concerning other groups of viruses. Notably, Hanko iflavirus-1 was especially prevalent in Culex eknomios (88.4%) and Ochlerotatus serratus (88.0%). Additionally, other ISVs, such as Guadalupe mosquito virus (GMV), Hubei mosquito virus1 (HMV1), Uxmal virus, Tanay virus, Cordoba virus, and Castlerea virus (all belonging to the Negevirus genus), were found as common viral species among the mosquitoes, although in lower proportions. These initial findings contribute to our understanding of ISVs within mosquito vectors of the Culicidae family in the Eastern Plains of Colombia. We recommend that future research explore deeper into ISV species shared among diverse vector species, and their potential interactions with arboviruses. In addition, we also showed the need for a thorough exploration of the influence of local rural habitat conditions on the shape of the virome in mosquito vectors.

The RNA landscape of Dunaliella salina in response to short-term salt stress.

Using the halotolerant green microalgae Dunaliella salina as a model organism has special merits, such as a wide range of salt tolerance, unicellular organism, and simple life cycle and growth conditions. These unique characteristics make it suitable for salt stress study. In order to provide an overview of the response of Dunaliella salina to salt stress and hopefully to reveal evolutionarily conserved mechanisms of photosynthetic organisms in response to salt stress, the transcriptomes and the genome of the algae were sequenced by the second and the third-generation sequencing technologies, then the transcriptomes under salt stress were compared to the transcriptomes under non-salt stress with the newly sequenced genome as the reference genome. The major cellular biological processes that being regulated in response to salt stress, include transcription, protein synthesis, protein degradation, protein folding, protein modification, protein transport, cellular component organization, cell redox homeostasis, DNA repair, glycerol synthesis, energy metabolism, lipid metabolism, and ion homeostasis. This study gives a comprehensive overview of how Dunaliella salina responses to salt stress at transcriptomic level, especially characterized by the nearly ubiquitous up-regulation of the genes involving in protein folding, DNA repair, and cell redox homeostasis, which may confer the algae important mechanisms to survive under salt stress. The three fundamental biological processes, which face huge challenges under salt stress, are ignored by most scientists and are worth further deep study to provide useful information for breeding economic important plants competent in tolerating salt stress, other than only depending on the commonly acknowledged osmotic balance and ion homeostasis.

New horizons of human genetics in digestive diseases

Recent studies have decoded the human Y chromosome sequencing with predominant precision and coverage, offering promising prospects in human genetics and clinical translation. Such an achievement is facilitated by third-generation...

LexicHash: Sequence Similarity Estimation via Lexicographic Comparison of Hashes.

Pairwise sequence alignment is a heavy computational burden, particularly in the context of third-generation sequencing technologies. This issue is commonly addressed by approximately estimating sequence similarities using a hash-based method such as MinHash. In MinHash, all k-mers in a read are hashed and the minimum hash value, the min-hash, is stored. Pairwise similarities can then be estimated by counting the number of min-hash matches between a pair of reads, across many distinct hash functions. The choice of the parameter k controls an important tradeoff in the task of identifying alignments: larger k-values give greater confidence in the identification of alignments (high precision) but can lead to many missing alignments (low recall), particularly in the presence of significant noise. In this work, we introduce LexicHash, a new similarity estimation method that is effectively independent of the choice of k and attains the high precision of large-k and the high sensitivity of small-k MinHash. LexicHash is a variant of MinHash with a carefully designed hash function. When estimating the similarity between two reads, instead of simply checking whether min-hashes match (as in standard MinHash), one checks how "lexicographically similar" the LexicHash min-hashes are. In our experiments on 40 PacBio datasets, the area under the Precision-Recall Curves obtained by LexicHash had an average improvement of 20.9% over MinHash. Additionally, the LexicHash framework lends itself naturally to an efficient search of the largest alignments, yielding an O(n) time algorithm, and circumventing the seemingly fundamental O(n2) scaling associated with pairwise similarity search. LexicHash is available on GitHub at https://github.com/gcgreenberg/LexicHash.

Characterization and phylogenetic analysis of the complete mitochondrial genome of a unique economic plant <i>Lycium barbarum</i> L.

<i>Lycium barbarum</i> L. (<i>L. barbarum</i>), an important medicinal and food crop from the Solanaceae family, is known for excellent tolerance to saline–alkaline and drought stresses. Therefore, it has significant research and application value. The nuclear and chloroplast genomes of <i>L. barbarum</i> have been previously reported; however, its mitochondrial genome is still unexplored. In this study, mitochondrial DNA sequencing and assembly in <i>L. barbarum</i> were studied via second- and third-generation sequencing technologies using the anthers collected from the main cultivars “Ningqi No.1” (fertile line) and its male-sterile natural mutant “Ningqi No.5” from Ningxia, China. The results revealed that “Ningqi No.1” and “Ningqi No.5” have the same mitochondrial circular genome structure with a total length of 413,881 bp. In total, 61 genes were annotated, including 35 protein-coding genes, four rRNA genes, 22 tRNA genes, and 2,242 open reading frames with unknown functions. The repeat sequences in mtDNA were analyzed, in which short repeats (30–59 bp) exhibited the highest number. Overall, 23 protein-coding genes in “Ningqi No.1” and “Ningqi No.5” exhibited RNA-editing phenomenon, with different RNA-editing site number and distribution position. Phylogenetic analysis demonstrated that <i>L. barbarum</i> has the closest relationship with <i>Hyoscyamus niger</i>. These results provided a basis for a comprehensive understanding of the mitochondrial genome of <i>L. barbarum</i> and the relevant data for omics studies on <i>L. barbarum</i>.

Differential adenine methylation analysis reveals increased variability in 6mA in the absence of methyl-directed mismatch repair.

Methylation greatly influences the bacterial genome by guiding DNA repair and regulating pathogenic and stress-response phenotypes. But, the rate of epigenetic changes and their consequences on molecular phenotypes are underexplored. Through a detailed characterization of genome-wide adenine methylation in a commonly used laboratory strain of Escherichia coli, we reveal that mismatch repair deficient populations experience an increase in epimutations resulting in a genome-wide reduction of 6mA methylation in a manner consistent with genetic drift. Our findings highlight how methylation patterns evolve and the constraints on epigenetic evolution due to post-replicative DNA repair, contributing to a deeper understanding of bacterial genome evolution and how epimutations may introduce semi-permanent variation that can influence adaptation.