Genomics in the long-read sequencing era.

Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.

HMMPolish: a coding region polishing tool for TGS-sequenced RNA viruses.

Access to accurate viral genomes is important to downstream data analysis. Third-generation sequencing (TGS) has recently become a popular platform for virus sequencing because of its long read length. However, its per-base error rate, which is higher than next-generation sequencing, can lead to genomes with errors. Polishing tools are thus needed to correct errors either before or after sequence assembly. Despite promising results of available polishing tools, there is still room to improve the error correction performance to perform more accurate genome assembly. The errors, particularly those in coding regions, can hamper analysis such as linage identification and variant monitoring. In this work, we developed a novel pipeline, HMMPolish, for correcting (polishing) errors in protein-coding regions of known RNA viruses. This tool can be applied to either raw TGS reads or the assembled sequences of the target virus. By utilizing profile Hidden Markov Models of protein families/domains in known viruses, HMMPolish can correct errors that are ignored by available polishers. We extensively validated HMMPolish on 34 datasets that covered four clinically important viruses, including HIV-1, influenza-A, norovirus, and severe acute respiratory syndrome coronavirus 2. These datasets contain reads with different properties, such as sequencing depth and platforms (PacBio or Nanopore). The benchmark results against popular/representative polishers show that HMMPolish competes favorably on error correction in coding regions of known RNA viruses.

Third-generation sequencing identified a novel complex variant in a patient with rare alpha-thalassemia.

BACKGROUND: Thalassemias represent some of the most common monogenic diseases worldwide and are caused by variations in human hemoglobin genes which disrupt the balance of synthesis between the alpha and beta globin chains. Thalassemia gene detection technology is the gold standard to achieve accurate detection of thalassemia, but in clinical practice, most of the tests are only for common genotypes, which can easily lead to missing or misdiagnosis of rare thalassemia genotypes. CASE PRESENTATION: We present the case of an 18-year-old Chinese female with abnormal values of routine hematological indices who was admitted for genetic screening for thalassemia. Genomic DNA was extracted and used for the genetic assays. Gap polymerase chain reaction and agarose gel electrophoresis were performed to detect HBA gene deletions, while PCR-reverse dot blot hybridization was used to detect point mutations in the HBA and HBB genes. Next-generation sequencing and third-generation sequencing (TGS) were used to identify known and potentially novel genotypes of thalassemia. We identified a novel complex variant αHb WestmeadαHb Westmeadαanti3.7/-α3.7 in a patient with rare alpha-thalassemia. CONCLUSIONS: Our study identified a novel complex variant that expands the thalassemia gene variants spectrum. Meanwhile, the study suggests that TGS could effectively improve the specificity of thalassemia gene detection, and has promising potential for the discovery of novel thalassemia genotypes, which could also improve the accuracy of genetic counseling. Couples who are thalassemia carriers have the opportunity to reduce their risk of having a child with thalassemia.

Third-generation sequencing: any future opportunities for PGT?

PURPOSE: To investigate use of the third-generation sequencing (TGS) Oxford Nanopore system as a new approach for preimplantation genetic testing (PGT). METHODS: Embryos with known structural variations underwent multiple displacement amplification to create fragments of DNA (average ~ 5 kb) suitable for sequencing on a nanopore. RESULTS: High-depth sequencing identified the deletion interval for the relatively large HBA1/2--SEA alpha thalassemia deletion. In addition, STRs were able to be identified in the primary sequence data for potential use in conventional PGT-M linkage confirmation. Sequencing of amplified embryo DNA carrying a translocation enabled balanced embryos to be identified and gave the precise identification of translocation breakpoints, offering the opportunity to differentiate carriers from non-carrier embryos. Low-pass sequencing gave reproducible profiles suitable for simple identification of whole-chromosome and segmental aneuploidies. CONCLUSION: TGS on the Oxford Nanopore is a possible alternative and versatile approach to PGT with potential for performing economical workups where the long read sequencing information can be used for assisting in a traditional PGT workup to design an accurate and reliable test. Additionally, application of TGS has the possibility of providing combined PGT-A/SR or in selected stand-alone PGT-M cases involving pathogenic deletions. Both of these applications offer the opportunity for simultaneous aneuploidy detection to select either balanced embryos for transfer or additional carrier identification. The low cost of the instrument offers new laboratories economical entry into onsite PGT.

Identification of a novel 107 kb deletion in the alpha-globin gene cluster using third-generation sequencing.

OBJECTIVE: To describe the characterization of a novel deletion causing α-thalassemia. METHODS: The proband, a 30-year-old female, displayed mild anemia from thalassemia screening. Gap-PCR was used to detect the four common deletional α-thalassemia, and a PCR-reverse dot blot was performed for the three point mutations of the α-globin gene. Multiplex ligation-dependent probe amplification (MLPA) was used to query possible breakpoints of a potential novel deletion. Third-generation sequencing (TGS) was used to identify the novel deletion after the MLPA failed. Gap-PCR and Sanger sequencing were validated for the breakpoint. RESULTS: No abnormal results were detected by Gap-PCR and PCR-reverse dot blot. MLPA only showed a large deletion upstream of the HBZ-1 probe, but the scope could not be determined. However, a novel 107 kb deletion at the α-globin gene was discovered by the TGS. The Gap-PCR products with the specific breakpoint fragment of the 107 kb deletion were confirmed by Sanger sequencing. CONCLUSIONS: A 107 kb deletion causing α-thalassemia was the first reported worldwide. TGS played an important role in this study and can be recommended as a reliable tool for rare or potential deletions in thalassemia.

Identification of double heterozygous -α4.2â /-α4.2â ¡ using third-generation sequencing.

OBJECTIVE: The 4.2 kb deletion (-α4.2/) is a common a+-thalassemia with a carrier rate, followed by the South-East Asian deletion (-SEA) and the 3.7 kb deletion (-α3.7/). There are few reports about 4.2 kb deletion sub-types. Herein, we present a patient with double heterozygous -α4.2Ⅰ/-α4.2Ⅱwho was identified using third-generation sequencing (TGS). METHODS: Hematology and hemoglobin fraction analysis were carried out by complete blood count (CBC) and capillary electrophoresis (CE). Gap-PCR was used to detect the common deletional α-thalassemia, and multiple ligation-dependent probe amplification (MLPA) was performed to screen the large deletion. Sanger sequencing identified the variant. The different deletions were confirmed by TGS. RESULTS: CBC showed the patient with microcytic hypochromic anemia, and CE indicated the presence of a Hb variant. Gap-PCR and MLPA detected 4.2 kb deletion homozygotes (-α4.2/-α4.2). The Hb variant was confirmed as Hb Q-Thailand by Sanger sequencing. The patient was identified as compound heterozygous of 4.2 kb deletion and Hb Q-Thailand (-α4.2/-α4.2-Q-Thailand, -α4.2Ⅰ/-α4.2Ⅱ) using TGS. CONCLUSIONS: Hb Q-Thailand (-α4.2-Q-Thailand/) complex 4.2 kb deletion heterozygote (-α4.2/) is easily misdiagnosed as 4.2 kb homozygous using Gap-PCR and MLPA. The TGS enables the identification of the two different 4.2 kb deletion sub-types.

Securing Group Patient Communication in 6G-Aided Dynamic Ubiquitous Healthcare with Real-Time Mobile DNA Sequencing.

(1) Background: With an advanced technique, third-generation sequencing (TGS) provides services with long deoxyribonucleic acid (DNA) reads and super short sequencing time. It enables onsite mobile DNA sequencing solutions for enabling ubiquitous healthcare (U-healthcare) services with modern mobile technology and smart entities in the internet of living things (IoLT). Due to some strict requirements, 6G technology can efficiently facilitate communications in a truly intelligent U-healthcare IoLT system. (2) Research problems: conventional single user-server architecture is not able to enable group conversations where "multiple patients-server" communication or "patient-patient" communication in the group is required. The communications are carried out via the open Internet, which is not a trusted channel. Since heath data and medical information are very sensitive, security and privacy concerns in the communication systems have become extremely important. (3) Purpose: the author aims to propose a dynamic group-based patient-authenticated key distribution protocol for 6G-aided U-healthcare services enabled by mobile DNA sequencing. In the protocol, an authenticated common session key is distributed by the server to the patients. Using the key, patients in a healthcare group are allowed to securely connect with the service provider or with each other for specific purposes of communication. (4) Results: the group key distribution process is protected by a secure three-factor authentication mechanism along with an efficient sequencing-device-based single sign-on (SD-SSO) solution. Based on traceable information stored in the server database, the proposed approach can provide patient-centered services which are available on multiple mobile devices. Security robustness of the proposed protocol is proven by well-known verification tools and a detailed semantic discussion. Performance evaluation shows that the protocol provides more functionality and incurs a reasonable overhead in comparison with the existing works.

Application of third-generation sequencing for genetic testing of thalassemia in Guizhou Province, Southwest China.

OBJECTIVES: To explore the application of third-generation sequencing (TGS) for genetic diagnosis and prenatal genetic screening of thalassemia genes. METHODS: Two groups of subjects were enrolled in this study. The first group included 176 subjects with positive hematological phenotypes for thalassemia. Thalassemia-associated genes were detected simultaneously in each sample using both the PacBio TGS platform based on single-molecule real-time (SMRT) technology and the conventional PCR-reverse dot blot (PCR-RDB). Sanger sequencing was used for validation when results were discordant between the two methods. The second group included 53 couples with at least one partner having a positive thalassemia hematological phenotype, and they were screened for homotypic thalassemia variants by TGS, and the risk of pregnancies with babies presenting with severe thalassemia, was assessed. RESULTS: Of the 176 subjects, 175 had concordant genotypes between the two methods, including 63 normal subjects and 112 α- and/or ß-thalassemia gene carriers, with a concordance rate of 99.43%. TGS detected a rare ß-thalassemia gene variant -50 (G > A) that was not detected by conventional PCR-RDB. TGS identified seven of the 53 couples as homotypic thalassemia gene carriers, five of whom were at risk of pregnancies with severe thalassemia. CONCLUSION: TGS could effectively detect common and rare thalassemia variants with high accuracy and efficiency. This approach would be suitable for prenatal thalassemia genetic screening in areas with high incidence of thalassemia.

Long-read Oxford nanopore sequencing reveals a de novo case of complex chromosomal rearrangement involving chromosomes 2, 7, and 13.

BACKGROUND: Complex chromosomal rearrangements (CCRs) are associated with high reproductive risk, infertility, abnormalities in offspring, and recurrent miscarriage in women. It is essential to accurately characterize apparently balanced chromosome rearrangements in unaffected individuals. METHODS: A CCR young couple who suffered two spontaneous abortions and underwent labor induction due to fetal chromosomal abnormalities was studied using long-read sequencing(LRS), single-nucleotide polymorphism (SNP) array, G-banding karyotype analysis (550-band resolution), and Sanger sequencing. RESULTS: SNP analysis of the amniotic fluid cells during the third pregnancy revealed a 9.9-Mb duplication at 7q21.11q21.2 and a 24.8-Mb heterozygous deletion at 13q21.1q31.1. The unaffected female partner was a carrier of a three-way CCR [46,XX,? ins(7;13)(q21.1;q21.1q22)t(2;13)(p23;q22)]. Subsequent LRS analysis revealed the exact breakpoint locations on the derivative chromosomes and the specific method of chromosome rearrangement, indicating that the CCR carrier was a more complex structural rearrangement comprising five breakpoints. Furthermore, LRS detected an inserted fragment of chromosome 13 in chromosome 7. CONCLUSIONS: LRS is effective for analyzing the complex structural variations of the human genome and may be used to clarify the specific CCRs for effective genetic counseling and appropriate intervention.

Analyzing Modern Biomolecules: The Revolution of Nucleic-Acid Sequencing - Review.

Recent developments have revolutionized the study of biomolecules. Among them are molecular markers, amplification and sequencing of nucleic acids. The latter is classified into three generations. The first allows to sequence small DNA fragments. The second one increases throughput, reducing turnaround and pricing, and is therefore more convenient to sequence full genomes and transcriptomes. The third generation is currently pushing technology to its limits, being able to sequence single molecules, without previous amplification, which was previously impossible. Besides, this represents a new revolution, allowing researchers to directly sequence RNA without previous retrotranscription. These technologies are having a significant impact on different areas, such as medicine, agronomy, ecology and biotechnology. Additionally, the study of biomolecules is revealing interesting evolutionary information. That includes deciphering what makes us human, including phenomena like non-coding RNA expansion. All this is redefining the concept of gene and transcript. Basic analyses and applications are now facilitated with new genome editing tools, such as CRISPR. All these developments, in general, and nucleic-acid sequencing, in particular, are opening a new exciting era of biomolecule analyses and applications, including personalized medicine, and diagnosis and prevention of diseases for humans and other animals.

The third generation sequencing: the advanced approach to genetic diseases.

Genomic sequencing technologies have revolutionized mutation detection of the genetic diseases in the past few years. In recent years, the third generation sequencing (TGS) has been gaining insight into more genetic diseases owing to the single molecular and real time sequencing technology. This paper reviews the genomic sequencing revolutionary history first and then focuses on the genetic diseases discovered through the TGS and the clinical effects of the TGS, which is followed by the discussion of the improvement in the bioinformatic analysis for the TGS and its limitations. In summary, the TGS has been enhancing the diagnostic accuracy of genetic diseases in molecular level as well as paving a new way for basic researches and therapies.

Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants.

Near full genome sequencing (NFGS) of HIV-1 is required to assess the genetic composition of HIV-1 strains comprehensively. Population-wide, it enables a determination of the heterogeneity of HIV-1 and the emergence of novel/recombinant strains, while for each individual it constitutes a diagnostic instrument to assist targeted therapeutic measures against viral components. There is still a lack of robust and adaptable techniques for efficient NFGS from miscellaneous HIV-1 subtypes. Using rational primer design, a broad primer set was developed for the amplification and sequencing of diverse HIV-1 group M variants from plasma. Using pure subtypes as well as diverse, unique recombinant forms (URF), variable amplicon approaches were developed for NFGS comprising all functional genes. Twenty-three different genomes composed of subtypes A (A1), B, F (F2), G, CRF01_AE, CRF02_AG, and CRF22_01A1 were successfully determined. The NFGS approach was robust irrespective of viral loads (≥306 copies/mL) and amplification method. Third-generation sequencing (TGS), single genome amplification (SGA), cloning, and bulk sequencing yielded similar outcomes concerning subtype composition and recombinant breakpoint patterns. The introduction of a simple and versatile near full genome amplification, sequencing, and cloning method enables broad application in phylogenetic studies of diverse HIV-1 subtypes and can contribute to personalized HIV therapy and diagnosis.

Comparative Genomics Provides Insights Into the Marine Adaptation in Sponge-Derived Kocuriaflava S43.

Sponge-derived actinomycetes represent a significant component of marine actinomycetes. Members of the genus Kocuria are distributed in various habitats such as soil, rhizosphere, clinical specimens, marine sediments, and sponges, however, to date, little is known about the mechanism of their environmental adaptation. Kocuria flava S43 was isolated from a coastal sponge. Phylogenetic analysis revealed that it was closely related to the terrestrial airborne K. flava HO-9041. In this study, to gain insights into the marine adaptation in K. flava S43 we sequenced the draft genome for K. flava S43 by third generation sequencing (TGS) and compared it with those of K. flava HO-9041 and some other Kocuria relatives. Comparative genomics and phylogenetic analyses revealed that K. flava S43 might adapt to the marine environment mainly by increasing the number of the genes linked to potassium homeostasis, resistance to heavy metals and phosphate metabolism, and acquiring the genes associated with electron transport and the genes encoding ATP-binding cassette (ABC) transporter, aquaporin, and thiol/disulfide interchange protein. Notably, gene acquisition was probably a primary mechanism of environmental adaptation in K. flava S43. Furthermore, this study also indicated that the Kocuria isolates from various marine and hyperosmotic environments possessed common genetic basis for environmental adaptation.

Assessment of Genetic Diversity and Structure of Large Garlic (Allium sativum) Germplasm Bank, by Diversity Arrays Technology "Genotyping-by-Sequencing" Platform (DArTseq).

Garlic (Allium sativum) is used worldwide in cooking and industry, including pharmacology/medicine and cosmetics, for its interesting properties. Identifying redundancies in germplasm blanks to generate core collections is a major concern, mostly in large stocks, in order to reduce space and maintenance costs. Yet, similar appearance and phenotypic plasticity of garlic varieties hinder their morphological classification. Molecular studies are challenging, due to the large and expected complex genome of this species, with asexual reproduction. Classical molecular markers, like isozymes, RAPD, SSR, or AFLP, are not convenient to generate germplasm core-collections for this species. The recent emergence of high-throughput genotyping-by-sequencing (GBS) approaches, like DArTseq, allow to overcome such limitations to characterize and protect genetic diversity. Therefore, such technology was used in this work to: (i) assess genetic diversity and structure of a large garlic-germplasm bank (417 accessions); (ii) create a core collection; (iii) relate genotype to agronomical features; and (iv) describe a cost-effective method to manage genetic diversity in garlic-germplasm banks. Hierarchical-cluster analysis, principal-coordinates analysis and STRUCTURE showed general consistency, generating three main garlic-groups, mostly determined by variety and geographical origin. In addition, high-resolution genotyping identified 286 unique and 131 redundant accessions, used to select a reduced size germplasm-bank core collection. This demonstrates that DArTseq is a cost-effective method to analyze species with large and expected complex genomes, like garlic. To the best of our knowledge, this is the first report of high-throughput genotyping of a large garlic germplasm. This is particularly interesting for garlic adaptation and improvement, to fight biotic and abiotic stresses, in the current context of climate change and global warming.