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Elbialy, Ali, M. A. El-Dosuky e Ibrahim M. El-Henawy. "Quality of Third Generation Sequencing". Journal of Computational and Theoretical Nanoscience 17, n. 12 (1 dicembre 2020): 5205–9. http://dx.doi.org/10.1166/jctn.2020.9630.
Testo completoAbstract (sommario):
Third generation sequencing (TGS) relates to long reads but with relatively high error rates. Quality of TGS is a hot topic, dealing with errors. This paper combines and investigates three quality related metrics. They are basecalling accuracy, Phred Quality Scores, and GC content. For basecalling accuracy, a deep neural network is adopted. The measured loss does not exceed 5.42.
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Hassan, Syahzuwan, Rosnah Bahar, Muhammad Farid Johan, Ezzeddin Kamil Mohamed Mohamed Hashim, Wan Zaidah Abdullah, Ezalia Esa, Faidatul Syazlin Abdul Abdul Hamid e Zefarina Zulkafli. "Next-Generation Sequencing (NGS) and Third-Generation Sequencing (TGS) for the Diagnosis of Thalassemia". Diagnostics 13, n. 3 (19 gennaio 2023): 373. http://dx.doi.org/10.3390/diagnostics13030373.
Testo completoAbstract (sommario):
Thalassemia is one of the most heterogeneous diseases, with more than a thousand mutation types recorded worldwide. Molecular diagnosis of thalassemia by conventional PCR-based DNA analysis is time- and resource-consuming owing to the phenotype variability, disease complexity, and molecular diagnostic test limitations. Moreover, genetic counseling must be backed-up by an extensive diagnosis of the thalassemia-causing phenotype and the possible genetic modifiers. Data coming from advanced molecular techniques such as targeted sequencing by next-generation sequencing (NGS) and third-generation sequencing (TGS) are more appropriate and valuable for DNA analysis of thalassemia. While NGS is superior at variant calling to TGS thanks to its lower error rates, the longer reads nature of the TGS permits haplotype-phasing that is superior for variant discovery on the homologous genes and CNV calling. The emergence of many cutting-edge machine learning-based bioinformatics tools has improved the accuracy of variant and CNV calling. Constant improvement of these sequencing and bioinformatics will enable precise thalassemia detections, especially for the CNV and the homologous HBA and HBG genes. In conclusion, laboratory transiting from conventional DNA analysis to NGS or TGS and following the guidelines towards a single assay will contribute to a better diagnostics approach of thalassemia.
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Chang, Wei-Fang, Shu-Min Chang, Pei-Ling Chu, Yi-Hsiu Chen, Rui-Hua Lee, Yi-Xuan Lee, Shun-Jen Tan et al. "#201 : Comparison of the Sensitivity of Detecting Cervical Bacteria with Next Generation Sequencing and Third Generation Sequencing Technologies". Fertility & Reproduction 05, n. 04 (dicembre 2023): 632–34. http://dx.doi.org/10.1142/s2661318223743631.
Testo completoAbstract (sommario):
Background and Aims: In in vitro fertilization (IVF) cycles, some patients suffering with vaginosis showed poor reproductive outcome even transferring with good quality embryos. The aim of this pilot study was to evaluate whether the species of Lactobacillus could be detected by next generation sequencing (NGS) and Third generation sequencing (TGS). Method: 18 Patients aged 32–48 years-old who visited our fertility center from February 2021 to June 2022 with previous failure of transfer cycle were included. Exclusion criteria was antibiotic treatment within 3 months prior to enrolment. Genomic DNA of cervical microbiota taken from Cervico vaginal swabs in all patients were extracted and amplified. NGS was performed following the protocol of Ion 16S Metagenomics Kit by detecting V2–4–8 and V3–6, 7–9 regions of 16S. Amplicons were sequenced with Ion GeneStudio S5 Prime System. For TGS, full length of 16S was sequenced with Single Molecule, Real-Time (SMRT) Sequencing (Pacific Biosciences) and analyzed. Results: All of the 18 cervical samples could be amplified with V2–4–8 and V3–6, 7–9 primers and the genus could be 100% identified by NGS. However, most of the Lactobacillus includes L. crispatus, L. jensenii. and L. gasseri showed indistinguishable except of L. iners. On the other hand, TGS clearly identified all Lactobacillus. For Gardnerella, Atopobium and Prevotella, TGS and NGS showed equivalent sensitivity in genus level. However, due to the sequence similarity, Escherichia/Shigella could not be identified from the Lactobacillus. Conclusion: In this report, we aim to compare the sensitivity of detecting bacteria by 16S amplification method. Preliminary results suggest that NGS can distinguish bacterium causes vaginosis in genus level, but there might be misleading if the existence of pathogen such as Escherichia/Shigella. Till now, TGS still exhibits the best sensitivity for distinguish Lactobacillus in species level.
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Scarano, Carmela, Iolanda Veneruso, Rosa Redenta De Simone, Gennaro Di Bonito, Angela Secondino e Valeria D’Argenio. "The Third-Generation Sequencing Challenge: Novel Insights for the Omic Sciences". Biomolecules 14, n. 5 (10 maggio 2024): 568. http://dx.doi.org/10.3390/biom14050568.
Testo completoAbstract (sommario):
The understanding of the human genome has been greatly improved by the advent of next-generation sequencing technologies (NGS). Despite the undeniable advantages responsible for their widespread diffusion, these methods have some constraints, mainly related to short read length and the need for PCR amplification. As a consequence, long-read sequencers, called third-generation sequencing (TGS), have been developed, promising to overcome NGS. Starting from the first prototype, TGS has progressively ameliorated its chemistries by improving both read length and base-calling accuracy, as well as simultaneously reducing the costs/base. Based on these premises, TGS is showing its potential in many fields, including the analysis of difficult-to-sequence genomic regions, structural variations detection, RNA expression profiling, DNA methylation study, and metagenomic analyses. Protocol standardization and the development of easy-to-use pipelines for data analysis will enhance TGS use, also opening the way for their routine applications in diagnostic contexts.
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Fukasawa, Yoshinori, Luca Ermini, Hai Wang, Karen Carty e Min-Sin Cheung. "LongQC: A Quality Control Tool for Third Generation Sequencing Long Read Data". G3: Genes|Genomes|Genetics 10, n. 4 (10 febbraio 2020): 1193–96. http://dx.doi.org/10.1534/g3.119.400864.
Testo completoAbstract (sommario):
We propose LongQC as an easy and automated quality control tool for genomic datasets generated by third generation sequencing (TGS) technologies such as Oxford Nanopore technologies (ONT) and SMRT sequencing from Pacific Bioscience (PacBio). Key statistics were optimized for long read data, and LongQC covers all major TGS platforms. LongQC processes and visualizes those statistics automatically and quickly.
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Athanasopoulou, Konstantina, Michaela A. Boti, Panagiotis G. Adamopoulos, Paraskevi C. Skourou e Andreas Scorilas. "Third-Generation Sequencing: The Spearhead towards the Radical Transformation of Modern Genomics". Life 12, n. 1 (26 dicembre 2021): 30. http://dx.doi.org/10.3390/life12010030.
Testo completoAbstract (sommario):
Although next-generation sequencing (NGS) technology revolutionized sequencing, offering a tremendous sequencing capacity with groundbreaking depth and accuracy, it continues to demonstrate serious limitations. In the early 2010s, the introduction of a novel set of sequencing methodologies, presented by two platforms, Pacific Biosciences (PacBio) and Oxford Nanopore Sequencing (ONT), gave birth to third-generation sequencing (TGS). The innovative long-read technologies turn genome sequencing into an ease-of-handle procedure by greatly reducing the average time of library construction workflows and simplifying the process of de novo genome assembly due to the generation of long reads. Long sequencing reads produced by both TGS methodologies have already facilitated the decipherment of transcriptional profiling since they enable the identification of full-length transcripts without the need for assembly or the use of sophisticated bioinformatics tools. Long-read technologies have also provided new insights into the field of epitranscriptomics, by allowing the direct detection of RNA modifications on native RNA molecules. This review highlights the advantageous features of the newly introduced TGS technologies, discusses their limitations and provides an in-depth comparison regarding their scientific background and available protocols as well as their potential utility in research and clinical applications.
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althomari, Moteab Abdulmohsen, Ibrahim Taher Bohassan, zahra hajji bohassan, Fayez Taher Alhajouji, Ebtihal Lafi M. Alhejaili, Talal Jubayr alharthi, Ahmed Mohammed Abdu Sofyani et al. "Thalassemia: Next Generation (NGS) and Third Generation Sequencing (TGS) for the Diagnosis." Egyptian Journal of Chemistry 67, n. 13 (1 dicembre 2024): 1519–31. https://doi.org/10.21608/ejchem.2024.336711.10811.
Testo completo
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Wong, Li Lian, Siti Aisyah Razali, Zulaikha Mat Deris, Muhd Danish-Daniel, Min Pau Tan, Siti Azizah Mohd Nor, Hongyu Ma et al. "Application of second-generation sequencing (SGS) and third generation sequencing (TGS) in aquaculture breeding program". Aquaculture 548 (febbraio 2022): 737633. http://dx.doi.org/10.1016/j.aquaculture.2021.737633.
Testo completo
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Notario, Elisabetta, Grazia Visci, Bruno Fosso, Carmela Gissi, Nina Tanaskovic, Maria Rescigno, Marinella Marzano e Graziano Pesole. "Amplicon-Based Microbiome Profiling: From Second- to Third-Generation Sequencing for Higher Taxonomic Resolution". Genes 14, n. 8 (31 luglio 2023): 1567. http://dx.doi.org/10.3390/genes14081567.
Testo completoAbstract (sommario):
The 16S rRNA amplicon-based sequencing approach represents the most common and cost-effective strategy with great potential for microbiome profiling. The use of second-generation sequencing (NGS) technologies has led to protocols based on the amplification of one or a few hypervariable regions, impacting the outcome of the analysis. Nowadays, comparative studies are necessary to assess different amplicon-based approaches, including the full-locus sequencing currently feasible thanks to third-generation sequencing (TGS) technologies. This study compared three different methods to achieve the deepest microbiome taxonomic characterization: (a) the single-region approach, (b) the multiplex approach, covering several regions of the target gene/region, both based on NGS short reads, and (c) the full-length approach, which analyzes the whole length of the target gene thanks to TGS long reads. Analyses carried out on benchmark microbiome samples, with a known taxonomic composition, highlighted a different classification performance, strongly associated with the type of hypervariable regions and the coverage of the target gene. Indeed, the full-length approach showed the greatest discriminating power, up to species level, also on complex real samples. This study supports the transition from NGS to TGS for the study of the microbiome, even if experimental and bioinformatic improvements are still necessary.
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Chen, Jiaqi, Qihui Chen, Huan Hu, Fang Wang, Xue Chen, Yang Zhang, Xiaoli Ma et al. "High-Accurate Third-Generation Sequencing to Comprehensively Decipher BCR::ABL1 TKIs in-Cis Resistant Mutations". Blood 144, Supplement 1 (5 novembre 2024): 3595. https://doi.org/10.1182/blood-2024-202681.
Testo completoAbstract (sommario):
Background Drug-resistant mutations in the ABL1 kinase domain (KD) of BCR::ABL1 are primary mechanisms of resistance to tyrosine kinase inhibitor (TKI) therapy in Ph-positive leukemia. These mutations alter the conformation of ABL1 within BCR::ABL1, impairing TKI and thus diminishing their anti-leukemia efficacy. Our previous studies, presented at the 61st and 62nd ASH Annual Meeting, demonstrated the superiority of next-generation sequencing (NGS) over Sanger sequencing for detecting BCR::ABL1-KD mutations. NGS offers higher sensitivity, accurate mutation frequency determination, and the ability to identify compound or polyclonal mutations within the same amplicon. However, NGS still needs to improve, including its short read length and the disadvantages in identifying in-cis compound mutations, lack of flexibility, and long turnaround time. Leveraging advancements in a novel third-generation sequencing (TGS) technology - characterized by single-molecule sequencing capability, long reads, real-time sequencing, and high accuracy - we have developed an approach for directly full-length in-cis BCR::ABL1-KD mutation screening. Methods The new approach allows for a comprehensive in-cis analysis of resistance mutations in the BCR::ABL1-KD using a novel TGS platform based on single-molecule side synthesis side nanopore sequencing (NSBS), distinct from PacBio or ONT sequencing. We retrospectively analyzed 30 specimens from 30 cases previously tested using NGS, including 15 mutation-positive and 15 mutation-negative specimens under previous investigation. Among them, there were ten single mutations, four double mutations, and one triple mutation, with the variant allele frequency (VAF) ranging from 7.8% to 99.6%. Results The TGS approach can directly analyze in-cis compound BCR::ABL1-KD mutations in a full-length BCR::ABL1-KD sequencing model with high single-base accuracy and superior to the NGS protocol. The novel TGS approach can detect the complete BCR::ABL1 fusion gene sequence (including p190 and p210 types, sequence range 1696~1717bp), which can more accurately obtain the ABL1 sequence in the BCR::ABL1 fusion gene, thus analyzing the veritable resistance mutations in the ABL1 KD of the BCR::ABL1 fusion gene; it has higher detection sensitivity (average sequencing coverage depth of 13086x, range 8226~24152x; Q30 can reach 99.39%; VAF as low as 1%). The TGS approach detected more mutations than NGS (27 vs. 21) and accurately distinguished more complex mutation patterns, including multiple compound or polyclonal mutations. It detected five mutations in the NGS mutation-negative group. The VAF obtained in mutation analysis was inconsistent compared to NGS, indicating that the mutations and mutation frequencies detected by TGS are more in line with the true situation and significant for continuous monitoring. Conclusion Through comparative analysis with the NGS project, the novel TGS approach shows the advantages of longer reads, higher sensitivity and accuracy, and the ability to complete the scheme from library construction to data output within 12 hours. It may provide the clinic with faster and more accurate results of in-cis BCR::ABL1-KD resistance mutation analysis with higher sensitivity and guide the clinic to quickly change treatment plans, thereby improving therapeutic outcomes.
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Thun, Gian Andri, Morgan Gueuning e Maja Mattle-Greminger. "Long-Read Sequencing in Blood Group Genetics". Transfusion Medicine and Hemotherapy 50, n. 3 (2023): 184–97. http://dx.doi.org/10.1159/000530652.
Testo completoAbstract (sommario):
Background: The key advantages of latest third-generation long-read sequencing (TGS) technologies include the ability to resolve long haplotypes and to characterize genomic regions that are challenging to analyze with short-read sequencing. Recent advancements in TGS technologies have significantly improved accuracy, a crucial requirement for the transition from research to diagnostic applications. Summary: In the field of immunohematology, the adoption of TGS is still in its early stages and published applications are scarce. An undeniable utility of TGS in blood group genomics is the ability to resolve ambiguous genotype-phenotype blood group results. In particular, hybrid genes and other large structural variants, as commonly found in the RHD/CE and MNS blood group systems, cause such discrepant results that can hardly be resolved by conventional methods. Long-read sequencing also greatly aids to generate high-standard reference alleles, establish haplotype sequence databases, or could even serve for high-resolution genotyping of all blood groups in parallel. Additionally, TGS holds the potential to close important knowledge gaps in blood group transcriptomics and epigenetics. Key Messages: The aims of this review were to examine the prospects of TGS technologies within the field of immunohematology and to highlight practical applications. Furthermore, we present a comprehensive overview of the existing and emerging wet-laboratory strategies for data generation, as well as a summary on bioinformatic data analysis methods. Finally, we provide an outlook on anticipated advancements in the near future.
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Ermini, Luca, e Patrick Driguez. "The Application of Long-Read Sequencing to Cancer". Cancers 16, n. 7 (25 marzo 2024): 1275. http://dx.doi.org/10.3390/cancers16071275.
Testo completoAbstract (sommario):
Cancer is a multifaceted disease arising from numerous genomic aberrations that have been identified as a result of advancements in sequencing technologies. While next-generation sequencing (NGS), which uses short reads, has transformed cancer research and diagnostics, it is limited by read length. Third-generation sequencing (TGS), led by the Pacific Biosciences and Oxford Nanopore Technologies platforms, employs long-read sequences, which have marked a paradigm shift in cancer research. Cancer genomes often harbour complex events, and TGS, with its ability to span large genomic regions, has facilitated their characterisation, providing a better understanding of how complex rearrangements affect cancer initiation and progression. TGS has also characterised the entire transcriptome of various cancers, revealing cancer-associated isoforms that could serve as biomarkers or therapeutic targets. Furthermore, TGS has advanced cancer research by improving genome assemblies, detecting complex variants, and providing a more complete picture of transcriptomes and epigenomes. This review focuses on TGS and its growing role in cancer research. We investigate its advantages and limitations, providing a rigorous scientific analysis of its use in detecting previously hidden aberrations missed by NGS. This promising technology holds immense potential for both research and clinical applications, with far-reaching implications for cancer diagnosis and treatment.
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Rohmer, Coralie, Hélène Touzet e Antoine Limasset. "Automated evaluation of multiple sequence alignment methods to handle third generation sequencing errors". PeerJ 12 (20 settembre 2024): e17731. http://dx.doi.org/10.7717/peerj.17731.
Testo completoAbstract (sommario):
Most third-generation sequencing (TGS) processing tools rely on multiple sequence alignment (MSA) methods to manage sequencing errors. Despite the broad range of MSA approaches available, a limited selection of implementations are commonly used in practice for this type of application, and no comprehensive comparative assessment of existing tools has been undertaken to date. In this context, we have developed an automatic pipeline, named MSA Limit, designed to facilitate the execution and evaluation of diverse MSA methods across a spectrum of conditions representative of TGS reads. MSA Limit offers insights into alignment accuracy, time efficiency, and memory utilization. It serves as a valuable resource for both users and developers, aiding in the assessment of algorithmic performance and assisting users in selecting the most appropriate tool for their specific experimental settings. Through a series of experiments using real and simulated data, we demonstrate the value of such exploration. Our findings reveal that in certain scenarios, popular methods may not consistently exhibit optimal efficiency and that the choice of the most effective method varies depending on factors such as sequencing depth, genome characteristics, and read error patterns. MSA Limit is an open source and freely available tool. All code and data pertaining to it and this manuscript are available at https://gitlab.cristal.univ-lille.fr/crohmer/msa-limit.
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Zhang, Wenjing, Neng Huang, Jiantao Zheng, Xingyu Liao, Jianxin Wang e Hong-Dong Li. "A Sequence-Based Novel Approach for Quality Evaluation of Third-Generation Sequencing Reads". Genes 10, n. 1 (14 gennaio 2019): 44. http://dx.doi.org/10.3390/genes10010044.
Testo completoAbstract (sommario):
The advent of third-generation sequencing (TGS) technologies, such as the Pacific Biosciences (PacBio) and Oxford Nanopore machines, provides new possibilities for contig assembly, scaffolding, and high-performance computing in bioinformatics due to its long reads. However, the high error rate and poor quality of TGS reads provide new challenges for accurate genome assembly and long-read alignment. Efficient processing methods are in need to prioritize high-quality reads for improving the results of error correction and assembly. In this study, we proposed a novel Read Quality Evaluation and Selection Tool (REQUEST) for evaluating the quality of third-generation long reads. REQUEST generates training data of high-quality and low-quality reads which are characterized by their nucleotide combinations. A linear regression model was built to score the quality of reads. The method was tested on three datasets of different species. The results showed that the top-scored reads prioritized by REQUEST achieved higher alignment accuracies. The contig assembly results based on the top-scored reads also outperformed conventional approaches that use all reads. REQUEST is able to distinguish high-quality reads from low-quality ones without using reference genomes, making it a promising alternative sequence-quality evaluation method to alignment-based algorithms.
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Fan, Xiaoying, Dong Tang, Yuhan Liao, Pidong Li, Yu Zhang, Minxia Wang, Fan Liang et al. "Single-cell RNA-seq analysis of mouse preimplantation embryos by third-generation sequencing". PLOS Biology 18, n. 12 (30 dicembre 2020): e3001017. http://dx.doi.org/10.1371/journal.pbio.3001017.
Testo completoAbstract (sommario):
The development of next generation sequencing (NGS) platform-based single-cell RNA sequencing (scRNA-seq) techniques has tremendously changed biological researches, while there are still many questions that cannot be addressed by them due to their short read lengths. We developed a novel scRNA-seq technology based on third-generation sequencing (TGS) platform (single-cell amplification and sequencing of full-length RNAs by Nanopore platform, SCAN-seq). SCAN-seq exhibited high sensitivity and accuracy comparable to NGS platform-based scRNA-seq methods. Moreover, we captured thousands of unannotated transcripts of diverse types, with high verification rate by reverse transcription PCR (RT-PCR)–coupled Sanger sequencing in mouse embryonic stem cells (mESCs). Then, we used SCAN-seq to analyze the mouse preimplantation embryos. We could clearly distinguish cells at different developmental stages, and a total of 27,250 unannotated transcripts from 9,338 genes were identified, with many of which showed developmental stage-specific expression patterns. Finally, we showed that SCAN-seq exhibited high accuracy on determining allele-specific gene expression patterns within an individual cell. SCAN-seq makes a major breakthrough for single-cell transcriptome analysis field.
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Diao, Shu, Xianying Ding, Qifu Luan e Jingmin Jiang. "A Complete Transcriptional Landscape Analysis of Pinus elliottii Engelm. Using Third-Generation Sequencing and Comparative Analysis in the Pinus Phylogeny". Forests 10, n. 11 (24 ottobre 2019): 942. http://dx.doi.org/10.3390/f10110942.
Testo completoAbstract (sommario):
The planting of Pinus elliottii Engelm. has now reached close to three million ha in China. Molecular breeding as part of the improvement program for P. elliottii in southern China has been carried out in recent years. Third-generation sequencing (Pacbio sequencing technology, TGS) was used to obtain the exome of P. elliottii for molecular breeding. A total of 35.8 Gb clean reads were generated using TGS. After removing the redundant reads, we obtained 80,339 high-accuracy transcripts. Significantly, a total of 76,411 transcripts (95.1%) were blasted to public annotation databases. We predicted 65,062 intact coding sequences (CDSs), 8916 alternative splicing events, 1937 long non-coding RNAs, and 22,109 simple sequence repeats (SSRs) based on these obtained transcripts. Using the public databases and the data obtained above, 23 orthologous single-copy genes were identified to analyze the phylogenetic relationships for Pinus firstly including P. elliottii. Many positive selection genes involved in important biological processes and metabolism pathways were identified between P. elliottii and other pines. These positive selection genes could be candidate genes to be researched on the genetic basis of superior performance. Our study is the first to reveal the full-length and well-annotated transcripts of P. elliottii, which could provide reference for short transcriptome sequences in the research of genetics, phylogenetics, and genetic improvement for the non-reference genome species.
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Cao, Ying, Haizhou Liu, Yi Yan, Wenjun Liu, Di Liu e Jing Li. "Discriminating Clonotypes of Influenza A Virus Genes by Nanopore Sequencing". International Journal of Molecular Sciences 22, n. 18 (17 settembre 2021): 10069. http://dx.doi.org/10.3390/ijms221810069.
Testo completoAbstract (sommario):
Influenza viruses still pose a serious threat to humans, and we have not yet been able to effectively predict future pandemic strains and prepare vaccines in advance. One of the main reasons is the high genetic diversity of influenza viruses. We do not know the individual clonotypes of a virus population because some are the majority and others make up only a small fraction of the population. First-generation (FGS) and next-generation sequencing (NGS) technologies have inherent limitations that are unable to resolve a minority clonotype’s information in the virus population. Third-generation sequencing (TGS) technologies with ultra-long reads have the potential to solve this problem but have a high error rate. Here, we evaluated emerging direct RNA sequencing and cDNA sequencing with the MinION platform and established a novel approach that combines the high accuracy of Illumina sequencing technology and long reads of nanopore sequencing technology to resolve both variants and clonotypes of influenza virus. Furthermore, a new program was written to eliminate the effect of nanopore sequencing errors for the analysis of the results. By using this pipeline, we identified 47 clonotypes in our experiment. We conclude that this approach can quickly discriminate the clonotypes of virus genes, allowing researchers to understand virus adaptation and evolution at the population level.
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Kim, Yoon-Jeon, You-Na Kim, Young-Hee Yoon, Eul-Ju Seo, Go-Hun Seo, Changwon Keum, Beom-Hee Lee e Joo-Yong Lee. "Diverse Genetic Landscape of Suspected Retinitis Pigmentosa in a Large Korean Cohort". Genes 12, n. 5 (30 aprile 2021): 675. http://dx.doi.org/10.3390/genes12050675.
Testo completoAbstract (sommario):
We conducted targeted next-generation sequencing (TGS) and/or whole exome sequencing (WES) to assess the genetic profiles of clinically suspected retinitis pigmentosa (RP) in the Korean population. A cohort of 279 unrelated Korean patients with clinically diagnosed RP and available family members underwent molecular analyses using TGS consisting of 88 RP-causing genes and/or WES with clinical variant interpretation. The combined genetic tests (TGS and/or WES) found a mutation in the 44 RP-causing genes and seven inherited retinal disease (IRD)-causing genes, and the total mutation detection rate was 57%. The mutation detection rate was higher in patients who experienced visual deterioration at a younger age (75.4%, age of symptom onset under 10 years) and who had a family history of RP (70.7%). The most common causative genes were EYS (8.2%), USH2A (6.8%), and PDE6B (4.7%), but mutations were dispersed among the 51 RP/IRD genes generally. Meanwhile, the PDE6B mutation was the most common in patients experiencing initial symptoms in their first decade, EYS in their second to third decades, and USH2A in their fifth decades and older. Of note, WES revealed some unexpected genotypes: ABCC6, CHM, CYP4V2, RS1, TGFBI, VPS13B, and WDR19, which were verified by ophthalmological re-phenotyping.
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Sahoo, Bikram, Sarwan Ali, Pin-Yu Chen, Murray Patterson e Alexander Zelikovsky. "Assessing the Resilience of Machine Learning Classification Algorithms on SARS-CoV-2 Genome Sequences Generated with Long-Read Specific Errors". Biomolecules 13, n. 6 (2 giugno 2023): 934. http://dx.doi.org/10.3390/biom13060934.
Testo completoAbstract (sommario):
The emergence of third-generation single-molecule sequencing (TGS) technology has revolutionized the generation of long reads, which are essential for genome assembly and have been widely employed in sequencing the SARS-CoV-2 virus during the COVID-19 pandemic. Although long-read sequencing has been crucial in understanding the evolution and transmission of the virus, the high error rate associated with these reads can lead to inadequate genome assembly and downstream biological interpretation. In this study, we evaluate the accuracy and robustness of machine learning (ML) models using six different embedding techniques on SARS-CoV-2 error-incorporated genome sequences. Our analysis includes two types of error-incorporated genome sequences: those generated using simulation tools to emulate error profiles of long-read sequencing platforms and those generated by introducing random errors. We show that the spaced k-mers embedding method achieves high accuracy in classifying error-free SARS-CoV-2 genome sequences, and the spaced k-mers and weighted k-mers embedding methods are highly accurate in predicting error-incorporated sequences. The fixed-length vectors generated by these methods contribute to the high accuracy achieved. Our study provides valuable insights for researchers to effectively evaluate ML models and gain a better understanding of the approach for accurate identification of critical SARS-CoV-2 genome sequences.
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Banin, Andrew N., Michael Tuen, Jude S. Bimela, Marcel Tongo, Paul Zappile, Alireza Khodadadi-Jamayran, Aubin J. Nanfack et al. "Development of a Versatile, Near Full Genome Amplification and Sequencing Approach for a Broad Variety of HIV-1 Group M Variants". Viruses 11, n. 4 (1 aprile 2019): 317. http://dx.doi.org/10.3390/v11040317.
Testo completoAbstract (sommario):
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.
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Zhou, Hui, Yu Sheng, Keli Qiu, Fei Ren, Pei Shi, Qingmei Xie, Jiying Guo, Haifa Pan e Jinyun Zhang. "Improved Annotation of the Peach (Prunus persica) Genome and Identification of Tissue- or Development Stage-Specific Alternative Splicing through the Integration of Iso-Seq and RNA-Seq Data". Horticulturae 9, n. 2 (30 gennaio 2023): 175. http://dx.doi.org/10.3390/horticulturae9020175.
Testo completoAbstract (sommario):
Alternative splicing (AS) is an important way to generate notable regulatory and proteomic complexity in eukaryotes. However, accurate full-length splicing isoform discovery by second-generation sequencing (SGS) technologies is beset with the precise assembly of multiple isoforms from the same gene loci. In recent years, third-generation sequencing (TGS) technologies have been adopted to gain insight into different aspects of transcriptome complexity, such as complete sequences of mRNA, alternative splicing, fusion transcript, and alternative polyadenylation (APA). Here, we combined PacBio Iso-Seq and Illumina RNA-Seq technologies to decipher the full-length transcriptome of peach. In total, 40,477 nonredundant high-quality consensus transcript sequences were obtained from equally pooled libraries from 10 samples of 6 organs, including leaf, shoot, flower, fruit peel, fruit mesocarp, and fruit stone, of which 18,274 isoforms were novel isoforms of known genes and 546 isoforms were novel gene transcripts. We also discovered 148 fusion transcripts, 15,434 AS events, 508 potential lncRNAs, and 4368 genes with APA events. Of these AS events, the most abundant (62.48%) AS type was intron retention (IR). Moreover, the expression levels of different isoforms identified in this study were quantitatively evaluated, and highly tissue- or development stage-specific expression patterns were observed. The novel transcript isoforms and new characteristics of the peach transcriptome revealed by this study will facilitate the annotation of the peach genome and lay the foundations for functional research in the future.
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Kawser, Zannat, Saikt Rahman, Emilie Westeel, Mohammad Tanbir Habib, Mohabbat Hossain, Md Rakibul Hassan Bulbul, Sharmin Aktar Mukta et al. "SARS-CoV-2 variant survey: Comparison of RT-PCR screening with TGS and variant distribution across two divisions of Bangladesh". PLOS ONE 19, n. 10 (17 ottobre 2024): e0311993. http://dx.doi.org/10.1371/journal.pone.0311993.
Testo completoAbstract (sommario):
Background The widespread increase in multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) since 2020 is causing significant health concerns worldwide. While whole-genome sequencing (WGS) has played a leading role in surveillance programs, many local laboratories lack the expertise and resources. Thus, we aimed to investigate the circulating SARS-CoV-2 variants and evaluate the performance of multiplexed real-time reverse transcription-PCR (RT-PCR) for screening and monitoring the emergence of new SARS-CoV-2 variants in Bangladesh. Methods A total of 600 confirmed SARS-CoV-2-positive cases were enrolled either prospectively or retrospectively from two divisions of Bangladesh. The samples were screened by variant RT-PCR targeting five mutations of the spike gene (N501Y, P681R, L452R, E484K, E484Q). A subsample of the study population was also selected for third-generation sequencing (TGS) and the results were compared to the variant RT-PCR screening. An in-depth comparison was made between the two methods in terms of congruence and cost-benefit. Result Seven variants were detected among samples, with similar distributions of the variants across both divisions. Variant RT-PCR for the targeted mutations lead to a 98.5% call rate; only nine samples failed to be determined. No association was found regarding the demographic features, clinical criteria, or routine RT-PCR Ct values across the variants. The clade diversity of the sequenced subpopulation (n = 99) exhibited similar distributions across the two study sites and other epidemiologic variables. Variant RT-PCR successfully distinguished variants of concern (VOCs) and variants of interest (VOIs); however, 8% discrepancy was observed for the closest lineages. Moreover, the variant RT-PCR represented an ideal balance of cost, time, and accuracy that outweigh their limitations. Conclusion Based on the strong agreement of variant RT-PCR with TGS, such rapid, easily accessible approaches of rapid strain typing are essential in the context of pandemic responses to guide both treatment decisions and public health measures.
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Le, Tuan-Vinh. "Cross-Server End-to-End Patient Key Agreement Protocol for DNA-Based U-Healthcare in the Internet of Living Things". Mathematics 11, n. 7 (28 marzo 2023): 1638. http://dx.doi.org/10.3390/math11071638.
Testo completoAbstract (sommario):
(1) Background: Third-generation sequencing (TGS) technique directly sequences single deoxyribonucleic acid (DNA) molecules, enabling real-time sequencing and reducing sequencing time from a few days to a few hours. Sequencing devices can be miniaturized and DNA-reading sensors placed on the body to monitor human health and vital signs, building an “internet of living things” (IoLT) facilitating ubiquitous healthcare services. In many cases, patients may wish to directly connect to each other for purposes of sharing real-time sequencing data, medical status or trading genomic data, etc. (2) Problems: User registration for a specific service may be limited due to some reason. Registering for multiple redundant services would also result in wasted money and possible wasteful communication overhead. In addition, since medical data and health information are very sensitive, security and privacy issues in the network are of paramount importance. (3) Methods: In this article, I propose a cross-server end-to-end (CS-E2E) patient authenticated key agreement protocol for DNA-based healthcare services in IoLT networks. My work allows two patients to mutually authenticate each other through assistance of respective servers, so that they can establish a reliable shared session key for securing E2E communications. The design employs multiple cost-saving solutions and robust cryptographic primitives, including smart-card-based single sign-on, elliptic curve cryptography, biohash function, etc. (4) Results: My proposed protocol is proven to be secure against various attacks and to incur reasonable communication cost compared to its predecessor works. The protocol also provides the support for more security properties and better functionalities. (5) Conclusions: The E2E communications between the patients are properly protected using the proposed approach. This assures a secure and efficient cross-server patient conversation for multiple purposes of healthcare communication.
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Le, Tuan-Vinh. "Securing Group Patient Communication in 6G-Aided Dynamic Ubiquitous Healthcare with Real-Time Mobile DNA Sequencing". Bioengineering 10, n. 7 (15 luglio 2023): 839. http://dx.doi.org/10.3390/bioengineering10070839.
Testo completoAbstract (sommario):
(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.
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Stamatopoulos, Basile, Adele Timbs, Hélène Dreau, Ruth Clifford, Pauline Robbe, Adam Burns, Joanne Mason e Anna Schuh. "Next-Generation Deep Sequencing Reveals Multiple Ighv Clones in One Third of CLL Patients Defining New Prognostic Subgroups and Improving Previous Classification". Blood 126, n. 23 (3 dicembre 2015): 4127. http://dx.doi.org/10.1182/blood.v126.23.4127.4127.
Testo completoAbstract (sommario):
Abstract Introduction: Chronic Lymphocytic Leukemia (CLL) is characterized by a heterogeneous clinical course. Currently, the mutational status of the immunoglobulin heavy chain variable (IgHV) region defines 2 risk groups: patients with ≥ 2% difference from the germline are considered as unmutated (UM) and have a poor prognosis while the opposite is observed for mutated (M) patients (Hamblin et al, 1999). Until now, clonality has been determined using PCR/gel electrophoresis and the percentage of IgHV mutations by Sanger sequencing using the Biomed-2 method (van Dongen, Leukemia 2003) which only allows for analysis of the major clone and assumes that only one clone per patient should be considered. Methods: in the present study, we sequenced the IgHV gene for 200 CLL patients with a median follow-up of 70 months (range, 1-309) by next-generation (NGS) and Sanger sequencing (SS) and investigated the impact on prognosis for the different subgroups. Briefly, 100ng of cDNA generated from RNA extracted from highly purified CD19+ cells obtained at diagnosis was amplified using IGH LEADER master mixes. For SS, clonality of PCR product was determined and monoclonal samples were directly sequenced. For NGS, PCR products were purified using magnetic beads, normalized and pooled to create a library for sequencing using the MiSeq v3 Reagent kit (600 cycles). Sequencing data was analyzed using LymphoTrack™ bioinformatics software and percentage of clone was based on the first 200 most abundant clones detected. Clones with an abundancy of < 2.5% were not considered. The IMGT database was used to calculate the percentage of mutation compared to germline. Results: Using SS, 49% (98/200) of patients were IgHV M, 38% (76/200) UM, while 13% (26/200) were polyclonal and further Sanger sequencing was inconclusive. Concordance between SS and NGS was 98.3%: for 90.2%, the major clone detected by NGS was the same by SS, for 8.5% the clone was the same but the percentage mutated compared to germ-line differed slightly with a median error of 0.6%, while for 1.7%, SS detected another clone which was less abundant by NGS. Interestingly, among patients considered as monoclonal by SS, 25.3% (44/174) were found to be polyclonal by NGS. NGS revealed that 35% (70/200) of patients display different IgHV re-arrangements in the same patient: the median frequency of the first two most abundant clones were 78.2% (range, 28.5-94.9) and 16.4% (range, 2.5-49.0) respectively. This implies that at least in these patients, leukemia-initiating events must occur prior to IgHV re-arrangement in a pro-B cell. Further in-depth studies of the HSC and early B cell progenitor compartments in these patients will be required to confirm these observations. In terms of prognosis, M, UM and polyclonal patients determined by SS had a median treatment-free survival (TFS) of 178, 29 and 129 months respectively (P<0.0001) and a median overall survival (OS) of >309, 183 and >309 months (P<0.0001). Since NGS was able to highlight different clones, we were able to create 5 different categories: patients with (a) multiple M clones, (b) 1 M clone, (c) a mix of M-UM clones, (d) 1 UM clone, (e) multiple UM clones. Using this new NGS classification, we found patients with different prognosis among patients already classified by SS. For example, SS-M patients were classified in 3 subgroups with a median TFS of >251 (a), 178 (b), 56 (c) months (P=0.0014). Similar results were observed for SS-UM patients who were divided in 3 subgroups with a median TFS of 94 (c), 24 (d), and 19 (e) month (P=0.0296). When all patients where considered, IgHV-NGS classification stratified patients in 5 different subgroups with median TFS of >251 (a), 178 (b), 57 (c), 24 (d), 19 (e) months (P<0.0001) and a median OS of 270, >309, >309, 183, 88 months (P<0.0001). Conclusions: determination of IgHV mutational status by NGS has excellent concordance with classical SS and in addition enables the detection of small clones which has a significant impact on prognosis. This allows the analysis of all clones without labor intensive manipulations especially for polyclonal patients. Here we showed for the first time that one third of CLL patients present with multiple IgHV subclones that impact on prognosis and refine the previous SS-IgHV classification. Figure 1. Figure 1. Disclosures Schuh: Acerta Pharma BV: Research Funding.
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Ge, Zang, Tan, Wang, Liu, Li, Wang, Chen, Zhan e Ma. "Single-Molecule Long-Read Sequencing of Avocado Generates Microsatellite Markers for Analyzing the Genetic Diversity in Avocado Germplasm". Agronomy 9, n. 9 (5 settembre 2019): 512. http://dx.doi.org/10.3390/agronomy9090512.
Testo completoAbstract (sommario):
Avocado (Persea americana Mill.) is an important fruit crop commercially grown in tropical and subtropical regions. Despite the importance of avocado, there is relatively little available genomic information regarding this fruit species. In this study, we functionally annotated the full-length avocado transcriptome sequence based on single-molecule real-time sequencing technology, and predicted the coding sequences (CDSs), transcription factors (TFs), and long non-coding RNA (lncRNA) sequences. Moreover, 76,777 simple sequence repeat (SSR) loci detected among the 42,096 SSR-containing transcript sequences were used to develop 149,733 expressed sequence tag (EST)-SSR markers. A subset of 100 EST-SSR markers was randomly chosen for an analysis that detected 15 polymorphicEST-SSR markers, with an average polymorphism information content of 0.45. These 15markers were able to clearly and effectively characterize46 avocado accessions based on geographical origin. In summary, our study is the first to generate a full-length transcriptome sequence and develop and analyze a set of EST-SSR markers in avocado. The application of third-generation sequencing techniques for developing SSR markers is a potentially powerful tool for genetic studies.
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Fan, Rong, Sabrina Bonde, Peng Gao, Brendan Sotomayor, Changya Chen, Tyler Mouw, Nicholas Zavazava e Kai Tan. "Dynamic HoxB4-regulatory network during embryonic stem cell differentiation to hematopoietic cells". Blood 119, n. 19 (10 maggio 2012): e139-e147. http://dx.doi.org/10.1182/blood-2011-12-396754.
Testo completoAbstract (sommario):
Abstract Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies to treat hematologic diseases. To date, HoxB4 remains the most effective transcription factor (TF) the overexpression of which in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop more efficient protocols for in vitro derivation of HSCs. In the present study, we performed global gene-expression profiling and ChIP coupled with deep sequencing at 4 stages of the HoxB4-mediated ESC differentiation toward HSCs. Joint analyses of ChIP/deep sequencing and gene-expression profiling unveiled several global features of the HoxB4 regulatory network. First, it is highly dynamic and gradually expands during the differentiation process. Second, HoxB4 functions as a master regulator of hematopoiesis by regulating multiple hematopoietic TFs and chromatin-modification enzymes. Third, HoxB4 acts in different combinations with 4 other hematopoietic TFs (Fli1, Meis1, Runx1, and Scl) to regulate distinct sets of pathways. Finally, the results of our study suggest that down-regulation of mitochondria and lysosomal genes by HoxB4 plays a role in the impaired lymphoid lineage development from ESC-derived HSCs.
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Nimma, Ramesh, Anil Kumar Kalvala, Nilkumar Patel, Sunil Kumar Surapaneni, Li Sun, Rakesh Singh, Ebony Nottingham et al. "Combined Transcriptomic and Proteomic Profiling to Unravel Osimertinib, CARP-1 Functional Mimetic (CFM 4.17) Formulation and Telmisartan Combo Treatment in NSCLC Tumor Xenografts". Pharmaceutics 14, n. 6 (28 maggio 2022): 1156. http://dx.doi.org/10.3390/pharmaceutics14061156.
Testo completoAbstract (sommario):
The epidermal growth factor receptor (EGFR) is highly expressed in many non-small cell lung cancers (NSCLC), necessitating the use of EGFR-tyrosine kinase inhibitors (TKIs) as first-line treatments. Osimertinib (OSM), a third-generation TKI, is routinely used in clinics, but T790M mutations in exon 20 of the EGFR receptor lead to resistance against OSM, necessitating the development of more effective therapeutics. Telmisartan (TLM), OSM, and cell cycle and apoptosis regulatory protein 1 (CARP-1) functional mimetic treatments (CFM4.17) were evaluated in this study against experimental H1975 tumor xenografts to ascertain their anti-cancer effects. Briefly, tumor growth was studied in H1975 xenografts in athymic nude mice, gene and protein expressions were analyzed using next-generation RNA sequencing, proteomics, RT-PCR, and Western blotting. TLM pre-treatment significantly reduced the tumor burden when combined with CFM-4.17 nanoformulation and OSM combination (TLM_CFM-F_OSM) than their respective single treatments or combination of OSM and TLM with CFM 4.17. Data from RNA sequencing and proteomics revealed that TLM_CFM-F_OSM decreased the expression of Lamin B2, STAT3, SOD, NFKB, MMP-1, TGF beta, Sox-2, and PD-L1 proteins while increasing the expression of AMPK proteins, which was also confirmed by RT-PCR, proteomics, and Western blotting. According to our findings, the TLM_CFM-F_OSM combination has a superior anti-cancer effect in the treatment of NSCLC by affecting multiple resistant markers that regulate mitochondrial homeostasis, inflammation, oxidative stress, and apoptosis.
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Upadhyaya, Hari D., Mahendar Thudi, Naresh Dronavalli, Neha Gujaria, Sube Singh, Shivali Sharma e Rajeev K. Varshney. "Genomic tools and germplasm diversity for chickpea improvement". Plant Genetic Resources 9, n. 01 (14 gennaio 2011): 45–58. http://dx.doi.org/10.1017/s1479262110000468.
Testo completoAbstract (sommario):
Chickpea is the third most important grain legume grown in the arid and semi-arid regions of the world. In spite of vast germplasm accessions available in different genebanks, there has been very limited use of these accessions in genetic enhancement of chickpea. However, in recent years, specialized germplasm subsets such as global composite collection, core collection, mini core collection and reference set have been developed. In parallel, significant genomic resources such as molecular markers including simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), diversity arrays technology (DArT) and transcript sequences, e.g. expressed sequence tags, short transcript reads, have been developed. By using SSR, SNP and DArT markers, integrated genetic maps have been developed. It is anticipated that the use of genomic resources and specialized germplasm subsets such as mini core collection and reference set will facilitate identification of trait-specific germplasm, trait mapping and allele mining for resistance to biotic and abiotic stresses and for agronomic traits. Advent of the next generation sequencing technologies coupled with advances in bioinformatics offers the possibility of undertaking large-scale sequencing of germplasm accessions so that modern breeding approaches such as genomic selection and breeding by design can be realized in near future for chickpea improvement.
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Kim, Taehyung, Joon Ho Moon, Yoo Jin Lee, Marc Tyndel, Jae-Sook Ahn, Hyeoung-Joon Kim, Yeo-Kyeoung Kim et al. "Longitudinal Tracking of MDS Patients Using Next Generation Sequencing Provides a Predictive Measure for Azacitidine Response and AML Progression". Blood 128, n. 22 (2 dicembre 2016): 52. http://dx.doi.org/10.1182/blood.v128.22.52.52.
Testo completoAbstract (sommario):
Abstract Introduction: Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders characterized by dysplastic changes in one or more cellular lineages causing impaired bone marrow function. One third of patients diagnosed with MDS progress to secondary acute myeloid leukemia (sAML). These patients have significantly worse prognoses than de novo AML patients. Azacitidine (AZA), a hypomethylating agent is commonly used to treat MDS patients as a frontline therapy. Although its survival benefits over supportive care in a randomized trial has been demonstrated, the underlying genetics and clonal dynamics upon AZA response/AML progression have not been well examined. Using next generation sequencing (NGS) technology, we attempted to assess the clinical relevance of somatic mutations and their dynamics as they relate to AZA treatment in MDS patients using longitudinal samples. Patients and Methods: Ninety-five MDS patients (56 lower risk and 39 higher risk MDS based on the revised IPSS scoring system) were enrolled in this study. The median age of the 95 patients is 67 years (range of 31 Ð 84) and median follow-up duration was 747 days (range of 137-3328 days). We performed targeted deep sequencing (entire exon region of a panel of 84 myeloid genes, Agilent custom probe set) on 285 bone-marrow samples including the longitudinal samples taken at diagnosis (n=95) and post-AZA treatment, (median 4 cycles) as well as T-cell fraction (CD3+). We multiplexed and sequenced the samples using an Illumina Hiseq 2000. After read mapping and variant calling, hierarchical clustering, pathway and survival analyses were performed in R. Results: Targeted sequencing on the myeloid gene panel revealed 176 mutations in 68 patients (68/95, 71.6%) with a median of 2 mutations per patient (ranges 2-6). The average on-target coverage for 285 sequenced samples was 1205x. Twenty-five of 44 mutated genes were recurrently mutated. ASXL1 was the most frequently mutated in the cohort (21%), followed by TET2 (15%), DNMT3A (11%), and SRSF2 (11%). Mutated genes were then grouped into 8 biological pathways, defined in The Cancer Genome Atlas (TCGA) AML study. The most frequent biological pathway with mutated genes at diagnosis was DNA methylation (28.4%), followed by spliceosome (25.2%), chromatin modifiers (22.1%), myeloid transcription factors (TFs) (11.6%), activated signaling (11.6%), tumor suppressors (12.6%), and cohesin complex (6.3%). When assessing the differences in patterns of variant allele frequency (VAF), we found significant VAF reduction in responders compared to non-responders (p = 0.007, repeated measures using general linear model, Figure A). Multivariate analyses revealed that mutation burden in different genes and biological pathways have distinct impact on AZA response, AML transformation, and overall survival. Higher bone marrow blast percentage (5%) was associated with all three measures (Figure B). Most significantly, mutations in activated signaling pathway genes are associated with AML progression (p=0.002). In addition, we could not detect decreased VAFs in activated signalling pathway genes even in responders (Figure C-D). Patients with SRSF2 mutations tend to respond to AZA (OR 14.084, p=0.003). Mutations in tumor suppressors (HR 4.825, p<0.001) and myeloid TFs (HR 3.070, p=0.020) were adverse prognostic factors in overall survival. Of interest, mutations in DNA methylation pathway were not independent prognostic factor for AZA response, AML transformation, or overall survival. Conclusion: These data and analyses show that reduction in mutation burden is correlated with AZA response. Mutations in different genes and biological pathways are associated with distinct clinical measures that tumor suppressors and myeloid TFs were identified as poor prognostic factors in terms of OS. Persistent mutation burden in activated signaling pathways is a strong predictor for AML transformation. In summary, longitudinal tracking of MDS patients using NGS may improve criteria for AZA response and early detection of AML progression. Figure 1. Figure 1. Disclosures Jang: Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Research Funding.
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Zang, He, Sijia Guo, Shunan Dong, Yuxuan Song, Kunze Li, Xiaoxue Fan, Jianfeng Qiu et al. "Construction of a Full-Length Transcriptome of Western Honeybee Midgut Tissue and Improved Genome Annotation". Genes 15, n. 6 (1 giugno 2024): 728. http://dx.doi.org/10.3390/genes15060728.
Testo completoAbstract (sommario):
Honeybees are an indispensable pollinator in nature with pivotal ecological, economic, and scientific value. However, a full-length transcriptome for Apis mellifera, assembled with the advanced third-generation nanopore sequencing technology, has yet to be reported. Here, nanopore sequencing of the midgut tissues of uninoculated and Nosema ceranae-inoculated A. mellifera workers was conducted, and the full-length transcriptome was then constructed and annotated based on high-quality long reads. Next followed improvement of sequences and annotations of the current reference genome of A. mellifera. A total of 5,942,745 and 6,664,923 raw reads were produced from midguts of workers at 7 days post-inoculation (dpi) with N. ceranae and 10 dpi, while 7,100,161 and 6,506,665 raw reads were generated from the midguts of corresponding uninoculated workers. After strict quality control, 6,928,170, 6,353,066, 5,745,048, and 6,416,987 clean reads were obtained, with a length distribution ranging from 1 kb to 10 kb. Additionally, 16,824, 17,708, 15,744, and 18,246 full-length transcripts were respectively detected, including 28,019 nonredundant ones. Among these, 43,666, 30,945, 41,771, 26,442, and 24,532 full-length transcripts could be annotated to the Nr, KOG, eggNOG, GO, and KEGG databases, respectively. Additionally, 501 novel genes (20,326 novel transcripts) were identified for the first time, among which 401 (20,255), 193 (13,365), 414 (19,186), 228 (12,093), and 202 (11,703) were respectively annotated to each of the aforementioned five databases. The expression and sequences of three randomly selected novel transcripts were confirmed by RT-PCR and Sanger sequencing. The 5′ UTR of 2082 genes, the 3′ UTR of 2029 genes, and both the 5′ and 3′ UTRs of 730 genes were extended. Moreover, 17,345 SSRs, 14,789 complete ORFs, 1224 long non-coding RNAs (lncRNAs), and 650 transcription factors (TFs) from 37 families were detected. Findings from this work not only refine the annotation of the A. mellifera reference genome, but also provide a valuable resource and basis for relevant molecular and -omics studies.
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Rienhoff, Hugh Y., Georges Natsoulis, Amber Jones, Jennifer Peppe, Ru Cao, Khalid Hanif e Justin M. Watts. "An Enhanced Sensitivity DNA Sequencing Protocol for the Detection in AML of Measurable Residual Disease (MRD) Applicable for All Mutations". Blood 132, Supplement 1 (29 novembre 2018): 5279. http://dx.doi.org/10.1182/blood-2018-99-115505.
Testo completoAbstract (sommario):
Abstract Background: One of the more important prognostic factors used to predict the outcome in acute myeloid leukemia (AML) is the persistence of leukemic cells after treatment. The reliable measurement of residual disease (MRD) offers many other clinical uses besides. An assay that was facile, affordable, and applicable to the broadest group of patients would find immediate favor. Next-generation sequencing (NGS) combined with various enrichment methods allow high sequencing depth on restricted targets. For instance, by enriching for <100 targets raw sequencing depths of 105 to 106 are routinely achieved. However, after excluding PCR duplicates identified using either end-point diversity of paired-end fragments or molecular tags, we found that NGS libraries effectively capture only a few percent of the input genomes (typically 1000 genomes or fewer for a 100ng input DNA corresponding to 30,000 genomes) effectively restricting the limit of detection (LOD) to approximately 1%. Hybrid capture and primer extension methods including protocols available from Nimblegen, Illumina for whole exome sequencing, exon capture using IDT probes and Nugen yielded similar results: all showed extensive loss of input genome diversity restricting the LOD (see Table 1 Section A). Most surprising was whole exome sequencing (WES). Sequencing 1000 ng input representing 300,000 genomes performed worst: for a given locus, loss of genomic diversity of approximately can be as high as 99.99% and is always more than 95% (Table 1A). More input is clearly not better. Methods and Results: We used the ThermoFisher multiplex PCR-based method to capture the 50 genomic fragments most frequently mutated in AML and labeled each with unique DNA tags. Sequence generated on an Ion Torrent showed that for 100ng of input DNA, >50% of the genomic diversity of that input DNA was preserved, i.e., >15,000 unique genomes were captured and sequenced from an initial 30,000 genomes resulting in a LOD of 0.1% with high precision as a mutation present at 0.1% in 15,000 genomes could be observed ~15 times. As shown in Table 1B, this method scales (non-linearly) as input DNA is increased; samples containing 500ng of DNA (~150,000 genomes) reproducibly permit an LOD of 0.01%, a sensitivity far exceeding any other non-allele specific method for measuring residual disease. In a real world application, we followed 3 AML patients from diagnosis to clinical CR. We tracked all mutations present at diagnosis using both IDT hybrid capture (HC) and the ThermoFisher (TF) method. One patient had molecular evidence of residual disease (VAF3%) that was detected and similarly quantitated by both HC and TF, in the second patient mutation were present at 1% by HC and 3% by TF . The TF value is the reliable one as it is derived from multiple independent templates. Finally the third patient was in molecular remission by HC but had a mutation allele detected by the TF with a frequency of 0.07%. We have extended these studies to include more than a dozen patients followed through relapse with similar results. These pilot studies provide clear evidence that standard sequencing methods cannot reliably promise LODs below 1%; further, the ThermoFisher method enhances the LOD at least 20-fold in an assay that can be applied to the great majority of AML patients. Table 1. Table 1. Disclosures Rienhoff: Imago BioSciences, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Natsoulis:Imago BioSciences, Inc.: Consultancy, Equity Ownership. Jones:Imago BioSciences, Inc.: Employment, Equity Ownership. Peppe:Imago BioSciences, Inc.: Employment, Equity Ownership. Cao:Thermo Fisher Scientific: Employment. Hanif:Thermo Fisher Scientific: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding.
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Rusk, Nicole. "Cheap third-generation sequencing". Nature Methods 6, n. 4 (aprile 2009): 244. http://dx.doi.org/10.1038/nmeth0409-244a.
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Schadt, E. E., S. Turner e A. Kasarskis. "A window into third-generation sequencing". Human Molecular Genetics 19, R2 (21 settembre 2010): R227—R240. http://dx.doi.org/10.1093/hmg/ddq416.
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Schadt, E. E., S. Turner e A. Kasarskis. "A window into third generation sequencing". Human Molecular Genetics 20, n. 4 (3 dicembre 2010): 853. http://dx.doi.org/10.1093/hmg/ddq481.
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Lee, Min-Sang, Hee Jin Cho, Jung Yong Hong, Jeeyun Lee, Se Hoon Park, Joon Oh Park, Young Suk Park et al. "Clinical and molecular distinctions in patients with refractory colon cancer who benefit from regorafenib treatment". Therapeutic Advances in Medical Oncology 12 (gennaio 2020): 175883592096584. http://dx.doi.org/10.1177/1758835920965842.
Testo completoAbstract (sommario):
Regorafenib (Stivarga, BAY 73-4506; Bayer Pharma AG, Berlin, Germany) is a novel oral multikinase inhibitor that blocks the activity of several protein kinases. However, few guidelines exist for novel biomarkers to select patients who will likely benefit from regorafenib treatment. Metastatic colorectal cancer (mCRC) patients treated with regorafenib were evaluated in this study. Tumor tissues of these patients were subjected to next-generation sequencing-based cancer panel tests. The relationship between molecular profiling and efficacy of regorafenib was analyzed. Among the 76 mCRC patients, the median age was 58 years (range 22–79 years), and 73.7% received regorafenib as a third-line therapy. The primary tumor locations were the right side ( n = 15, 19.8%) and the left side ( n = 61, 80.2%). Most patients (97.4%) had received prior anti-angiogenetic agents, and a prior anti-Epidermal Growth Factor Receptor (EGFR) agent had been administered to 32.9%. Of these 76 patients, 65 were evaluated to determine the efficacy of treatment. We observed zero complete responses, seven confirmed partial responses (PR 9.2%), 26 stable disease states (34.2%), and 32 disease progressions (42.1%). The overall confirmed response rate and the disease control rate were 9.2% and 43.4%, respectively. Genomic analysis revealed that APC mutations were significant in patients who demonstrated a tumor response to regorafenib ( p < 0.05). Interestingly, FGFR1 amplification was detected in only three of 76 patients (3.9%), and these three patients achieved a PR to regorafenib. The median progression-free survival time was 2.8 months (95% Confidence Interval [CI] 1.6–4.0). Patients with BRAF mutation and/or SMAD4 mutation had significantly worse progression-free survival (PFS) than those without such a mutation. On pathway analysis, Tumor Growth Factor (TGF)-beta pathways were significantly associated with worse PFS. We found that efficacy of regorafenib might be correlated with specific genetic aberrations, such as APC mutation and FGFR1 amplification. In addition, SMAD4 mutation and TGF-beta pathway were associated with worse PFS after regorafenib. We found that efficacy of regorafenib might be correlated with specific genetic aberrations, such as APC mutation and FGFR1 amplification. In addition, SMAD4 mutation and the TGF-beta pathway were associated with worse PFS after regorafenib.
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Levine, Jonathan, Sydney Finkelstein e Venkata Arun Timmaraju. "Analyzing imbalance of short tandem repeats for pancreatic cancer detection." Journal of Clinical Oncology 42, n. 16_suppl (1 giugno 2024): e16359-e16359. http://dx.doi.org/10.1200/jco.2024.42.16_suppl.e16359.
Testo completoAbstract (sommario):
e16359 Background: Short tandem repeats (STRs), alternatively known as microsatellites, encompass repeat units of 1 to 6 base pairs (bp) in the genome and function as regulators of gene expression, impacting plasticity and complex traits. STRs are prone to hypermutation with alterations correlated to pathogenicity in multiple Mendelian and acquired disorders. STR variant classes include insertions, mobile element insertions (MEIs), deletions, and multi-allelic copy number variants (mCNVs). These can lead to repeat expansions, allelic imbalance (AI), and microsatellite instability (MSI) observed in diseases such as Huntington's disease, hereditary ataxia, and multiple cancers. In pancreatic cancer, AI and associated loss-of-heterozygosity (LOH) at tumor suppressor loci flanked by hypervariable STR regions correspond to a pathogenic phenotype with a risk of aggressive disease. Consequently, it is critical to develop accurate and robust methods to precisely identify and genotype pancreatic samples with varying specimen quality in a clinical setting to aid in diagnosis. Methods: Capillary electrophoresis (CE) and Sanger sequencing have been used to analyze STRs. The availability of newer tools such as 2nd generation short-read (NGS) and 3rd generation long-read (TGS) sequencers could allow for high throughput scaling, however, informatics challenges associated with analyzing repetitive regions remain. Here we developed amplification and enrichment methods for targeted analysis for detection by CE, NGS and TGS. Results: Amplification and enrichment strategies for detecting 17 short tandem repeat regions that play a role in pancreatic carcinogenesis were developed. Methods were optimized for detecting STR alterations in cell-free nucleic acids isolated from endoscope-guided pancreatic fine needle aspirate biopsies. Furthermore, informatics tools were developed to analyze short-read and long-read sequencing data to accurately analyze repeat regions. Conclusions: Here we compared 3 platforms (CE, NGS, and TGS) and found that CE remains the gold standard for analyzing AI/LOH with data comparable to long-read TGS, while short-read NGS is not always amenable to accurate analysis dependent on the region of interest. Additionally, we found that artifacts of amplification persist, irrespective of the 3 platforms analyzed.
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Chen, Xizhen. "Progress in the Application of CRISPR/Cas Family Mediated Third-generation Sequencing Technology". Academic Journal of Science and Technology 12, n. 2 (14 settembre 2024): 184–90. http://dx.doi.org/10.54097/a43yhk04.
Testo completoAbstract (sommario):
The first generation sequencing technology based on the dideoxynucleotide (ddNTP) chain termination method proposed by Sanger was gradually eliminated due to its high cost, low sequencing read length and cumbersome process. The second generation of sequencing technology, called High-throughput sequencing (HTS), which was then developed, still has the problem of fixed read length. In recent years, the third generation sequencing technology represented by SMRT technology and Nanopore sequencing technology has gradually become popular. Compared with the previous two generations of sequencing technology, the most significant advantage of the third generation sequencing technology is its ability to carry out single molecule sequencing. In this process, the infinite length of nucleic acid sequence can be determined theoretically without the help of PCR amplification. This paper first introduces the basic principles, advantages and disadvantages of third-generation sequencing, and introduces in detail the CRISPR/Cas family-mediated SMRT technology and Nanopore sequencing technology in the third-generation sequencing technology. Finally, the research progress and prospects of the combination of the third generation sequencing technology and gene editing technology in the future are analyzed.
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Munroe, David J., e Timothy J. R. Harris. "Third-generation sequencing fireworks at Marco Island". Nature Biotechnology 28, n. 5 (maggio 2010): 426–28. http://dx.doi.org/10.1038/nbt0510-426.
Testo completo
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Chen, Zhiao, e Xianghuo He. "Application of third-generation sequencing in cancer research". Medical Review 1, n. 2 (21 ottobre 2021): 150–71. http://dx.doi.org/10.1515/mr-2021-0013.
Testo completoAbstract (sommario):
Abstract In the past several years, nanopore sequencing technology from Oxford Nanopore Technologies (ONT) and single-molecule real-time (SMRT) sequencing technology from Pacific BioSciences (PacBio) have become available to researchers and are currently being tested for cancer research. These methods offer many advantages over most widely used high-throughput short-read sequencing approaches and allow the comprehensive analysis of transcriptomes by identifying full-length splice isoforms and several other posttranscriptional events. In addition, these platforms enable structural variation characterization at a previously unparalleled resolution and direct detection of epigenetic marks in native DNA and RNA. Here, we present a comprehensive summary of important applications of these technologies in cancer research, including the identification of complex structure variants, alternatively spliced isoforms, fusion transcript events, and exogenous RNA. Furthermore, we discuss the impact of the newly developed nanopore direct RNA sequencing (RNA-Seq) approach in advancing epitranscriptome research in cancer. Although the unique challenges still present for these new single-molecule long-read methods, they will unravel many aspects of cancer genome complexity in unprecedented ways and present an encouraging outlook for continued application in an increasing number of different cancer research settings.
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Dumschott, Kathryn, Maximilian H.-W. Schmidt, Harmeet Singh Chawla, Rod Snowdon e Björn Usadel. "Oxford Nanopore sequencing: new opportunities for plant genomics?" Journal of Experimental Botany 71, n. 18 (27 maggio 2020): 5313–22. http://dx.doi.org/10.1093/jxb/eraa263.
Testo completoAbstract (sommario):
Abstract DNA sequencing was dominated by Sanger’s chain termination method until the mid-2000s, when it was progressively supplanted by new sequencing technologies that can generate much larger quantities of data in a shorter time. At the forefront of these developments, long-read sequencing technologies (third-generation sequencing) can produce reads that are several kilobases in length. This greatly improves the accuracy of genome assemblies by spanning the highly repetitive segments that cause difficulty for second-generation short-read technologies. Third-generation sequencing is especially appealing for plant genomes, which can be extremely large with long stretches of highly repetitive DNA. Until recently, the low basecalling accuracy of third-generation technologies meant that accurate genome assembly required expensive, high-coverage sequencing followed by computational analysis to correct for errors. However, today’s long-read technologies are more accurate and less expensive, making them the method of choice for the assembly of complex genomes. Oxford Nanopore Technologies (ONT), a third-generation platform for the sequencing of native DNA strands, is particularly suitable for the generation of high-quality assemblies of highly repetitive plant genomes. Here we discuss the benefits of ONT, especially for the plant science community, and describe the issues that remain to be addressed when using ONT for plant genome sequencing.
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Ueda, Yoshih ide. "New sequencing technology, third‐generation nanopore sequencing, for hepatitis C virus research". Hepatology Research 49, n. 9 (settembre 2019): 961–63. http://dx.doi.org/10.1111/hepr.13383.
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Gao, Longlong, Wenjie Xu, Tianyi Xin e Jingyuan Song. "Application of third-generation sequencing to herbal genomics". Frontiers in Plant Science 14 (7 marzo 2023). http://dx.doi.org/10.3389/fpls.2023.1124536.
Testo completoAbstract (sommario):
There is a long history of traditional medicine use. However, little genetic information is available for the plants used in traditional medicine, which limits the exploitation of these natural resources. Third-generation sequencing (TGS) techniques have made it possible to gather invaluable genetic information and develop herbal genomics. In this review, we introduce two main TGS techniques, PacBio SMRT technology and Oxford Nanopore technology, and compare the two techniques against Illumina, the predominant next-generation sequencing technique. In addition, we summarize the nuclear and organelle genome assemblies of commonly used medicinal plants, choose several examples from genomics, transcriptomics, and molecular identification studies to dissect the specific processes and summarize the advantages and disadvantages of the two TGS techniques when applied to medicinal organisms. Finally, we describe how we expect that TGS techniques will be widely utilized to assemble telomere-to-telomere (T2T) genomes and in epigenomics research involving medicinal plants.
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Pei, Surui, Tao Liu, Xue Ren, Weizhong Li, Chongjian Chen e Zhi Xie. "Benchmarking variant callers in next-generation and third-generation sequencing analysis". Briefings in Bioinformatics, 23 luglio 2020. http://dx.doi.org/10.1093/bib/bbaa148.
Testo completoAbstract (sommario):
Abstract DNA variants represent an important source of genetic variations among individuals. Next- generation sequencing (NGS) is the most popular technology for genome-wide variant calling. Third-generation sequencing (TGS) has also recently been used in genetic studies. Although many variant callers are available, no single caller can call both types of variants on NGS or TGS data with high sensitivity and specificity. In this study, we systematically evaluated 11 variant callers on 12 NGS and TGS datasets. For germline variant calling, we tested DNAseq and DNAscope modes from Sentieon, HaplotypeCaller mode from GATK and WGS mode from DeepVariant. All the four callers had comparable performance on NGS data and 30× coverage of WGS data was recommended. For germline variant calling on TGS data, we tested DNAseq mode from Sentieon, HaplotypeCaller mode from GATK and PACBIO mode from DeepVariant. All the three callers had similar performance in SNP calling, while DeepVariant outperformed the others in InDel calling. TGS detected more variants than NGS, particularly in complex and repetitive regions. For somatic variant calling on NGS, we tested TNscope and TNseq modes from Sentieon, MuTect2 mode from GATK, NeuSomatic, VarScan2, and Strelka2. TNscope and Mutect2 outperformed the other callers. A higher proportion of tumor sample purity (from 10 to 20%) significantly increased the recall value of calling. Finally, computational costs of the callers were compared and Sentieon required the least computational cost. These results suggest that careful selection of a tool and parameters is needed for accurate SNP or InDel calling under different scenarios.
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Huang, Weilun, Shoufang Qu, Qiongzhen Qin, Xu Yang, Wanqing Han, Yongli Lai, Jiaqi Chen, Shihao Zhou, Xuexi Yang e Wanjun Zhou. "Nanopore Third-Generation Sequencing for Comprehensive Analysis of Hemoglobinopathy Variants". Clinical Chemistry, 14 giugno 2023. http://dx.doi.org/10.1093/clinchem/hvad073.
Testo completoAbstract (sommario):
Abstract Background Oxford Nanopore Technology (ONT) third-generation sequencing (TGS) is a versatile genetic diagnostic platform. However, it is nonetheless challenging to prepare long-template libraries for long-read TGS, particularly the ONT method for analysis of hemoglobinopathy variants involving complex structures and occurring in GC-rich and/or homologous regions. Methods A multiplex long PCR was designed to prepare library templates, including the whole-gene amplicons for HBA2/1, HBG2/1, HBD, and HBB, as well as the allelic amplicons for targeted deletions and special structural variations. Library construction was performed using long-PCR products, and sequencing was conducted on an Oxford Nanopore MinION instrument. Genotypes were identified based on integrative genomics viewer (IGV) plots. Results This novel long-read TGS method distinguished all single nucleotide variants and structural variants within HBA2/1, HBG2/1, HBD, and HBB based on the whole-gene sequence reads. Targeted deletions and special structural variations were also identified according to the specific allelic reads. The result of 158 α-/β-thalassemia samples showed 100% concordance with previously known genotypes. Conclusions This ONT TGS method is high-throughput, which can be used for molecular screening and genetic diagnosis of hemoglobinopathies. The strategy of multiplex long PCR is an efficient strategy for library preparation, providing a practical reference for TGS assay development.
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Traisrisilp, Kuntharee, Yu Zheng, Kwong Wai Choy e Pimlak Chareonkwan. "Thalassemia screening by third-generation sequencing: Pilot study in a Thai population". Obstetric Medicine, 26 ottobre 2023. http://dx.doi.org/10.1177/1753495x231207676.
Testo completoAbstract (sommario):
Background Conventional thalassemia screening takes a stepwise approach and has limitations in comprehensively identifying all spectrums of mutations. This study aimed to investigate the performance of third-generation sequencing (TGS) compared to conventional molecular testing. Methods TGS was applied to validate all known variants detected by conventional testing and to detect missing variants in undiagnosed cases. The study was conducted at Maharaj Nakorn Chiang Mai Hospital between December 2021 and April 2022. Results In total, 19 cases were included in this study, among which 52.6% (10/19) had known thalassemia variants, while 47.7% (9/19) cases were undiagnosed by conventional methods. All 16 variants previously detected were validated by TGS, and TGS additionally detected 43.8% (7/16) thalassemia variants for 36.8% (7/19) cases. Conclusion TGS could provide additional genetic diagnoses compared with conventional methods. Further cost-effectiveness studies with a larger sample size are needed to explore the role of TGS in clinical practices.
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Du, Nan, Jiayu Shang e Yanni Sun. "Improving protein domain classification for third-generation sequencing reads using deep learning". BMC Genomics 22, n. 1 (9 aprile 2021). http://dx.doi.org/10.1186/s12864-021-07468-7.
Testo completoAbstract (sommario):
Abstract Background With the development of third-generation sequencing (TGS) technologies, people are able to obtain DNA sequences with lengths from 10s to 100s of kb. These long reads allow protein domain annotation without assembly, thus can produce important insights into the biological functions of the underlying data. However, the high error rate in TGS data raises a new challenge to established domain analysis pipelines. The state-of-the-art methods are not optimized for noisy reads and have shown unsatisfactory accuracy of domain classification in TGS data. New computational methods are still needed to improve the performance of domain prediction in long noisy reads. Results In this work, we introduce ProDOMA, a deep learning model that conducts domain classification for TGS reads. It uses deep neural networks with 3-frame translation encoding to learn conserved features from partially correct translations. In addition, we formulate our problem as an open-set problem and thus our model can reject reads not containing the targeted domains. In the experiments on simulated long reads of protein coding sequences and real TGS reads from the human genome, our model outperforms HMMER and DeepFam on protein domain classification. Conclusions In summary, ProDOMA is a useful end-to-end protein domain analysis tool for long noisy reads without relying on error correction.
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Zhan, Lixia, Chunrong Gui, Wei Wei, Juliang Liu e Baoheng Gui. "Third generation sequencing transforms the way of the screening and diagnosis of thalassemia: a mini-review". Frontiers in Pediatrics 11 (6 luglio 2023). http://dx.doi.org/10.3389/fped.2023.1199609.
Testo completoAbstract (sommario):
Thalassemia is an inherited blood disorder imposing a significant social and economic burden. Comprehensive screening strategies are essential for the prevention and management of this disease. Third-generation sequencing (TGS), a breakthrough technology, has shown great potential for screening and diagnostic applications in various diseases, while its application in thalassemia detection is still in its infancy. This review aims to understand the latest and most widespread uses, advantages of TGS technologies, as well as the challenges and solutions associated with their incorporation into routine screening and diagnosis of thalassemia. Overall, TGS has exhibited higher rates of positive detection and diagnostic accuracy compared to conventional methods and next-generation sequencing technologies, indicating that TGS will be a feasible option for clinical laboratories conducting in-house thalassemia testing. The implementation of TGS technology in thalassemia diagnosis will facilitate the development of effective prevention and management strategies, thereby reducing the burden of this disease on individuals and society.
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Wang, Ying, Xiaohan Li, Wenxiang Lu, Fuyu Li, Lingsong Yao, Zhiyu Liu, Hua-Juan Shi, Weizhong Zhang e Yunfei Bai. "Full-length circRNA sequencing method using low-input RNA and profiling of circRNAs in MPTP-PD mice on nanopore platform". Analyst, 2024. http://dx.doi.org/10.1039/d4an00715h.
Testo completoAbstract (sommario):
Considering the importance of accurate information of full-length (FL) transcripts in functional analysis, researchers prefer to develop new sequencing methods base on third-generation sequencing (TGS) rather than short-read sequencing. Several...
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Zhang, Lu, FengXin Chen, Zhan Zeng, Mengjiao Xu, Fangfang Sun, Liu Yang, Xiaoyue Bi et al. "Advances in Metagenomics and Its Application in Environmental Microorganisms". Frontiers in Microbiology 12 (17 dicembre 2021). http://dx.doi.org/10.3389/fmicb.2021.766364.
Testo completoAbstract (sommario):
Metagenomics is a new approach to study microorganisms obtained from a specific environment by functional gene screening or sequencing analysis. Metagenomics studies focus on microbial diversity, community constitute, genetic and evolutionary relationships, functional activities, and interactions and relationships with the environment. Sequencing technologies have evolved from shotgun sequencing to high-throughput, next-generation sequencing (NGS), and third-generation sequencing (TGS). NGS and TGS have shown the advantage of rapid detection of pathogenic microorganisms. With the help of new algorithms, we can better perform the taxonomic profiling and gene prediction of microbial species. Functional metagenomics is helpful to screen new bioactive substances and new functional genes from microorganisms and microbial metabolites. In this article, basic steps, classification, and applications of metagenomics are reviewed.