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Komarova, Natalia, Daria Barkova et Alexander Kuznetsov. « Implementation of High-Throughput Sequencing (HTS) in Aptamer Selection Technology ». International Journal of Molecular Sciences 21, no 22 (20 novembre 2020) : 8774. http://dx.doi.org/10.3390/ijms21228774.
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Aptamers are nucleic acid ligands that bind specifically to a target of interest. Aptamers have gained in popularity due to their high potential for different applications in analysis, diagnostics, and therapeutics. The procedure called systematic evolution of ligands by exponential enrichment (SELEX) is used for aptamer isolation from large nucleic acid combinatorial libraries. The huge number of unique sequences implemented in the in vitro evolution in the SELEX process imposes the necessity of performing extensive sequencing of the selected nucleic acid pools. High-throughput sequencing (HTS) meets this demand of SELEX. Analysis of the data obtained from sequencing of the libraries produced during and after aptamer isolation provides an informative basis for precise aptamer identification and for examining the structure and function of nucleic acid ligands. This review discusses the technical aspects and the potential of the integration of HTS with SELEX.
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Pérez-Losada, Marcos, Miguel Arenas, Juan Carlos Galán, Mª Alma Bracho, Julia Hillung, Neris García-González et Fernando González-Candelas. « High-throughput sequencing (HTS) for the analysis of viral populations ». Infection, Genetics and Evolution 80 (juin 2020) : 104208. http://dx.doi.org/10.1016/j.meegid.2020.104208.
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He, Xuejun, Ningzhi Zhang, Wenye Cao, Yiqiao Xing et Ning Yang. « Application Progress of High-Throughput Sequencing in Ocular Diseases ». Journal of Clinical Medicine 11, no 12 (17 juin 2022) : 3485. http://dx.doi.org/10.3390/jcm11123485.
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Ocular diseases affect multiple eye parts and can be caused by pathogenic infections, complications of systemic diseases, genetics, environment, and old age. Understanding the etiology and pathogenesis of eye diseases and improving their diagnosis and treatment are critical for preventing any adverse consequences of these diseases. Recently, the advancement of high-throughput sequencing (HTS) technology has paved wide prospects for identifying the pathogenesis, signaling pathways, and biomarkers involved in eye diseases. Due to the advantages of HTS in nucleic acid sequence recognition, HTS has not only identified several normal ocular surface microorganisms but has also discovered many pathogenic bacteria, fungi, parasites, and viruses associated with eye diseases, including rare pathogens that were previously difficult to identify. At present, HTS can directly sequence RNA, which will promote research on the occurrence, development, and underlying mechanism of eye diseases. Although HTS has certain limitations, including low effectiveness, contamination, and high cost, it is still superior to traditional diagnostic methods for its efficient and comprehensive diagnosis of ocular diseases. This review summarizes the progress of the application of HTS in ocular diseases, intending to explore the pathogenesis of eye diseases and improve their diagnosis.
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Aronesty, Erik. « Comparison of Sequencing Utility Programs ». Open Bioinformatics Journal 7, no 1 (31 janvier 2013) : 1–8. http://dx.doi.org/10.2174/1875036201307010001.
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High throughput sequencing (HTS) has resulted in extreme growth rates of sequencing data. At our lab, we generate terabytes of data every day. It is usually seen as required for data output to be “cleaned” and processed in various ways prior to use for common tasks such as variant calling, expression quantification and assembly. Two common tasks associated with HTS are adapter trimming and paired-end joining. I have developed two tools at Expression Analysis, Inc. to address these common tasks. The names of these programs are fastq-mcf and fastq-join. I compared the performance of these tools to similar open-source utilities, both in terms of resource efficiency, and effectiveness.
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Pu, Dan, et Pengfeng Xiao. « A real-time decoding sequencing technology—new possibility for high throughput sequencing ». RSC Advances 7, no 64 (2017) : 40141–51. http://dx.doi.org/10.1039/c7ra06202h.
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GIZA, ALEKSANDRA, EWELINA IWAN, ARKADIUSZ BOMBA et DARIUSZ WASYL. « Basics of high throughput sequencing Summary ». Medycyna Weterynaryjna 77, no 11 (2025) : 6589–2025. http://dx.doi.org/10.21521/mw.6594.
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Sequencing can provide genomic characterisation of a specific organism, as well as of a whole environmental or clinical sample. High Throughput Sequencing (HTS) makes it possible to generate an enormous amount of genomic data at gradually decreasing costs and almost in real-time. HTS is used, among others, in medicine, veterinary medicine, microbiology, virology and epidemiology. The paper presents practical aspects of the HTS technology. It describes generations of sequencing, which vary in throughput, read length, accuracy and costs ̶ and thus are used for different applications. The stages of HTS, as well as their purposes and pitfalls, are presented: extraction of the genetic material, library preparation, sequencing and data processing. For success of the whole process, all stages need to follow strict quality control measurements. Choosing the right sequencing platform, proper sample and library preparation procedures, as well as adequate bioinformatic tools are crucial for high quality results.
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Malapi-Wight, Martha, Bishwo Adhikari, Jing Zhou, Leticia Hendrickson, Clarissa J. Maroon-Lango, Clint McFarland, Joseph A. Foster et Oscar P. Hurtado-Gonzales. « HTS-Based Diagnostics of Sugarcane Viruses : Seasonal Variation and Its Implications for Accurate Detection ». Viruses 13, no 8 (17 août 2021) : 1627. http://dx.doi.org/10.3390/v13081627.
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Rapid global germplasm trade has increased concern about the spread of plant pathogens and pests across borders that could become established, affecting agriculture and environment systems. Viral pathogens are of particular concern due to their difficulty to control once established. A comprehensive diagnostic platform that accurately detects both known and unknown virus species, as well as unreported variants, is playing a pivotal role across plant germplasm quarantine programs. Here we propose the addition of high-throughput sequencing (HTS) from total RNA to the routine quarantine diagnostic workflow of sugarcane viruses. We evaluated the impact of sequencing depth needed for the HTS-based identification of seven regulated sugarcane RNA/DNA viruses across two different growing seasons (spring and fall). Our HTS analysis revealed that viral normalized read counts (RPKM) was up to 23-times higher in spring than in the fall season for six out of the seven viruses. Random read subsampling analyses suggested that the minimum number of reads required for reliable detection of RNA viruses was 0.5 million, with a viral genome coverage of at least 92%. Using an HTS-based total RNA metagenomics approach, we identified all targeted viruses independent of the time of the year, highlighting that higher sequencing depth is needed for the identification of DNA viruses.
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Bester, Rachelle, Chanel Steyn, Johannes H. J. Breytenbach, Rochelle de Bruyn, Glynnis Cook et Hans J. Maree. « Reproducibility and Sensitivity of High-Throughput Sequencing (HTS)-Based Detection of Citrus Tristeza Virus and Three Citrus Viroids ». Plants 11, no 15 (26 juillet 2022) : 1939. http://dx.doi.org/10.3390/plants11151939.
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The credibility of a pathogen detection assay is measured using specific parameters including repeatability, specificity, sensitivity, and reproducibility. The use of high-throughput sequencing (HTS) as a routine detection assay for viruses and viroids in citrus was previously evaluated and, in this study, the reproducibility and sensitivity of the HTS assay were assessed. To evaluate the reproducibility of HTS, the same plants assayed in a previous study were sampled again, one year later, and assessed in triplicate using the same analyses to construct the virome profile. The sensitivity of the HTS assay was compared to routinely used RT-PCR assays in a time course experiment, to compensate for natural pathogen accumulation in plants over time. The HTS pipeline applied in this study produced reproducible and comparable results to standard RT-PCR assays for the detection of CTV and three viroid species in citrus. Even though the limit of detection of HTS can be influenced by pathogen concentration, sample processing method and sequencing depth, detection with HTS was found to be either equivalent or more sensitive than RT-PCR in this study.
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Bester, Rachelle, Glynnis Cook et Hans J. Maree. « Citrus Tristeza Virus Genotype Detection Using High-Throughput Sequencing ». Viruses 13, no 2 (23 janvier 2021) : 168. http://dx.doi.org/10.3390/v13020168.
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The application of high-throughput sequencing (HTS) has successfully been used for virus discovery to resolve disease etiology in many agricultural crops. The greatest advantage of HTS is that it can provide a complete viral status of a plant, including information on mixed infections of viral species or virus variants. This provides insight into the virus population structure, ecology, or evolution and can be used to differentiate among virus variants that may contribute differently toward disease etiology. In this study, the use of HTS for citrus tristeza virus (CTV) genotype detection was evaluated. A bioinformatic pipeline for CTV genotype detection was constructed and evaluated using simulated and real data sets to determine the parameters to discriminate between false positive read mappings and true genotype-specific genome coverage. A 50% genome coverage cut-off was identified for non-target read mappings. HTS with the associated bioinformatic pipeline was validated and proposed as a CTV genotyping assay.
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Kunej, Urban, Aida Dervishi, Valérie Laucou, Jernej Jakše et Nataša Štajner. « The Potential of HTS Approaches for Accurate Genotyping in Grapevine (Vitis vinifera L.) ». Genes 11, no 8 (10 août 2020) : 917. http://dx.doi.org/10.3390/genes11080917.
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The main challenge associated with genotyping based on conventional length polymorphisms is the cross-laboratory standardization of allele sizes. This step requires the inclusion of standards and manual sizing to avoid false results. Capillary electrophoresis (CE) approaches limit the information to the length polymorphism and do not allow the determination of a complete marker sequence. As an alternative, high-throughput sequencing (HTS) offers complete information regarding marker sequences and their flanking regions. In this work, we investigated the suitability of a semi-quantitative sequencing approach for microsatellite genotyping using Illumina paired-end technology. Twelve microsatellite loci that are well established for grapevine CE typing were analysed on 96 grapevine samples from six different countries. We redesigned primers to the length of the amplicon for short sequencing (~100 bp). The primer pair was flanked with a 10 bp overhang for the introduction of barcodes on both sides of the amplicon to enable high multiplexing. The highest data peaks were determined as simple sequence repeat (SSR) alleles and compared with the CE dataset based on 12 reference samples. The comparison showed that HTS SSR genotyping can successfully replace the CE system in further experiments. We believe that, with next-generation sequencing, genotyping can be improved in terms of its speed, accuracy, and price.
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WANG, MINGBANG, XIAOMEI FAN, TAO WANG et JINYU WU. « High-throughput sequencing of autism spectrum disorders comes of age ». Genetics Research 95, no 4 (août 2013) : 121–29. http://dx.doi.org/10.1017/s0016672313000153.
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SummaryAutism spectrum disorders (ASDs) are lifelong neurodevelopmental disabilities that affect 1 in 88 children in the USA. Despite the high heritability, the genetic basis for a majority of the ASDs remains elusive. The considerable clinical and genetic heterogeneity pose a significant challenge technically. State-of-the-art high-throughput sequencing (HTS), which makes the analyses of any specific single/multiple genes or whole exomes feasible, has shown a promising perspective in disease gene discovery. To date, numerous genetic studies using HTS have been reported and many rare inherited or de novo mutations have been identified. This review will focus on the progress and prospective of genome studies of ASDs using HTS.
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Fuentes, Azahara, Alicia Serrano, Blanca Ferrer Lores, Veronica Lendinez, Carolina Monzo, Carmen Ivorra, Mar Tormo, Maria Jose Terol, Blanca Navarro et Javier F. Chaves. « Ighv Mutational Status By Deep Next Generation Sequencing Refines Ighv Sanger Sequencing Classification in Patients with Chronic Lymphocytic Leukaemia ». Blood 134, Supplement_1 (13 novembre 2019) : 3028. http://dx.doi.org/10.1182/blood-2019-129145.
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Introduction: Determination of the mutational status of rearranged immunoglobulin heavy chain variable (IgHV) genes in patients with Chronic Lymphocytic Leukaemia (CLL), is considered one of the most important prognostic factors: patients with unmutated IgHV (UM; ≥98% of identity to the germline) genes have a more aggressive disease course and develop more frequently unfavourable genetic deletions or mutations than patients with mutated IgHV (M; ≤98%). Mutational status, is currently determined by Sanger sequencing (Sseq) that allows the analysis of the major clone, however, international guidelines recommend caution in assigning mutational status in cases with "Borderline" IgHV identity (97-97.9%), and cases with double rearrangements with discordant mutational status. Objective: Analyze and determine the mutational status of the IgHV locus by High-throughput sequencing (HTS), in a cohort of CLL patients (n=51) with unclassifiable Sseq results: borderline status (n=22); double rearrangements (n=27) with discordant mutational status (n=2). Methods: We included 51 DNA samples extracted from peripheral blood of patients diagnosed of CLL according to the National Cancer Institute Working Group guidelines in our institution between 1986 and 2019 (median absolute lymphocytes 11.4x109/L [2,8-239,5x109/L]). Sseq amplification and analysis of IgHV rearrangements were performed on DNA conforming to the updated ERIC recommendations. In all the cases we were able to determinate the IGVH identity. To switch high-throughput sequencing to the clinical practice, we assessed the reliability of different library preparation methods to sequence IGH locus in patients with CLL. Amplification was performed using the Sequencing Multiplex Kit based on IGH FR (forward primers) and consensus JH (reverse primer) multiplex. PCR products were purified using Magsi-NGS Prep magnetic beads (Magnamedics Diagnostics), normalized and pooled to create a library for sequencing using a MiSeq equipment. To simplify and make automatic the analysis of the same we developed a specific bioinformatic pipeline that covers from preprocessing to final data summarization and interpretation. The backbone of the analysis includes read preprocessing, mapping against IMGT reference sequences, consensus IgHV reads pairwise alignment to determine mutational status and read classification into rearrangements. Results: This approach led to the identification of a dominant clone IgHV in all cases (n=51). Instead, the percentage of identity calculated by HTS analysis varies in: - 15/22 borderline cases whose mutational status could be recalculated into 10 MM and 5 UM. The rest 7 remaining in borderline group. - We could identify both clones in 29 double rearrangements cases, with concordant mutational status except 2/29 undetermined cases, included in UM group regarding HTS results. Our tool led to the identification of a dominant clonotypic IgHV in all cases, and when compared the HTS sequence/mutational status for the most abundant clone with Sseq and for the IgHV status determination, 15 out of 22 (68,18%), could be reclassified. This case showed a major clone with productive rearrangement mutated by Sseq but unmutated by HTS. Conclusions: Analyze and determine the mutational status of the IgHV locus by HTS, would potentially reveal multiple rearrangements and increase the prognostic precision of IgHV mutation analysis. IgHV-HTS classification is able to precisely classify patients with borderline status or/and multiple IgHV rearrangements for which Sseq is inconclusive. In this case, it has been possible to improved prognostication for 17 out of 24 patients. This is helping us to discover the advantages of the data obtained by HTS compared with current Sseq standard technique. Samples were provided by the INCLIVA Biobank. Funded by Gilead Felowship 257/17 Disclosures Terol: Abbvie: Consultancy; Janssen: Consultancy, Research Funding; Gilead: Research Funding; Roche: Consultancy; Astra Zeneca: Consultancy.
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GIZA, ALEKSANDRA, EWELINA IWAN et DARIUSZ WASYL. « Application of high throughput sequencing in veterinary science ». Medycyna Weterynaryjna 78, no 02 (2022) : 6622–2022. http://dx.doi.org/10.21521/mw.6622.
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High throughput sequencing (HTS) creates an opportunity for comprehensive genomic studies. It can be applied in veterinary science, bacteriology and virology, diagnostics of animal diseases, food safety, examinations of the composition of environmental samples, and even in veterinary vaccinology. Thus HTS a wide-ranging method that can be applied in different areas of the One Health approach. In particular, the whole genome sequencing (WGS) of bacteria is routinely used in food hygiene and outbreak investigations for phylogenetic analysis of pathogenic bacteria isolated from various sources across timeline, molecular characterisation of bacteria, plasmids, antibiotic resistance and identification of virulence factors. Metagenomics can be used to characterize the composition of microbiota in environmental samples. It makes it possible to obtain a taxonomic identification of bacteria, fungi or plants present in a metasample. It can also be used for the monitoring and epidemiological tracing of viruses, such as SARS-CoV-2. The transcriptomic approach makes it possible to study the expression of genes associated with various infections and diseases. HTS is a highly versatile method, but the selection of the proper application is crucial to obtain expected outcomes. The paper presents some HTS approaches and examples of research in veterinary science.
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Ness, Tara E., Andrew DiNardo et Maha R. Farhat. « High Throughput Sequencing for Clinical Tuberculosis : An Overview ». Pathogens 11, no 11 (14 novembre 2022) : 1343. http://dx.doi.org/10.3390/pathogens11111343.
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High throughput sequencing (HTS) can identify the presence of Mycobacterium tuberculosis DNA in a clinical sample while also providing information on drug susceptibility. Multiple studies have provided a context for exploring the clinical application of HTS for TB diagnosis. The workflow challenges, strengths and limitations of the various sequencing platforms, and tools used for analysis are presented to provide a framework for further innovations in the field.
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Villamor, D. E. V., T. Ho, M. Al Rwahnih, R. R. Martin et I. E. Tzanetakis. « High Throughput Sequencing For Plant Virus Detection and Discovery ». Phytopathology® 109, no 5 (mai 2019) : 716–25. http://dx.doi.org/10.1094/phyto-07-18-0257-rvw.
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Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists’ utility in their research.
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Boegel, Sebastian, John C. Castle et Andreas Schwarting. « Current status of use of high throughput nucleotide sequencing in rheumatology ». RMD Open 7, no 1 (janvier 2021) : e001324. http://dx.doi.org/10.1136/rmdopen-2020-001324.
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ObjectiveHere, we assess the usage of high throughput sequencing (HTS) in rheumatic research and the availability of public HTS data of rheumatic samples.MethodsWe performed a semiautomated literature review on PubMed, consisting of an R-script and manual curation as well as a manual search on the Sequence Read Archive for public available HTS data.ResultsOf the 699 identified articles, rheumatoid arthritis (n=182 publications, 26%), systemic lupus erythematous (n=161, 23%) and osteoarthritis (n=152, 22%) are among the rheumatic diseases with the most reported use of HTS assays. The most represented assay is RNA-Seq (n=457, 65%) for the identification of biomarkers in blood or synovial tissue. We also find, that the quality of accompanying clinical characterisation of the sequenced patients differs dramatically and we propose a minimal set of clinical data necessary to accompany rheumatological-relevant HTS data.ConclusionHTS allows the analysis of a broad spectrum of molecular features in many samples at the same time. It offers enormous potential in novel personalised diagnosis and treatment strategies for patients with rheumatic diseases. Being established in cancer research and in the field of Mendelian diseases, rheumatic diseases are about to become the third disease domain for HTS, especially the RNA-Seq assay. However, we need to start a discussion about reporting of clinical characterisation accompany rheumatological-relevant HTS data to make clinical meaningful use of this data.
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Glasa, Miroslav, Katarína Šoltys, Lukáš Predajňa, Nina Sihelská, Jaroslav Budiš, Michaela Mrkvová, Ján Kraic, Daniel Mihálik et Ana Belén Ruiz-García. « High-throughput sequencing of Potato virus M from tomato in Slovakia reveals a divergent variant of the virus ». Plant Protection Science 55, No. 3 (17 mai 2019) : 159–66. http://dx.doi.org/10.17221/144/2018-pps.
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High-throughput sequencing (HTS) analysis of tomato (Solanum lycopersicum) samples revealed the presence of Potato virus M (PVM) in this crop in Slovakia. Full-length genomes of three PVM isolates were obtained using both HTS and Sanger sequencing validation. While two isolates (T40 and T50) were shown to belong to major Group I, a divergent T20 isolate was phylogenetically unrelated to any known PVM variant, potentially representing a new phylogenetic group. Despite a relatively high intraspecies diversity (17.3 ± 0.3%), no evidence of recombination was detected in the dataset of available complete PVM sequences. Conventional screening of tomato plants in Slovakia using ELISA and RT-PCR further confirmed a frequent occurrence of PVM in this host. Developed RT-PCR showed its polyvalence to detect the PVM Group I isolates, however, in silico analysis of primer binding sites indicated its compromised use for Group II isolates. Our results further pinpoint the significance of HTS for unbiased unveiling of virus diversity and a need for continual optimisation of molecular detection tools.
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Kurtz, David M., Michael R. Green, Scott V. Bratman, Florian Scherer, Chih Long Liu, Christian A. Kunder, Kazuhiro Takahashi et al. « Noninvasive monitoring of diffuse large B-cell lymphoma by immunoglobulin high-throughput sequencing ». Blood 125, no 24 (11 juin 2015) : 3679–87. http://dx.doi.org/10.1182/blood-2015-03-635169.
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Key Points DLBCL can be detected in the blood by immunoglobulin high-throughput sequencing (Ig-HTS) with high specificity. Although DLBCL can be detected in leukocytes or plasma by Ig-HTS, plasma has greater sensitivity and more accurately reflects disease.
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Javaran, Vahid Jalali, Peter Moffett, Pierre Lemoyne, Dong Xu, Charith Raj Adkar-Purushothama et Mamadou Lamine Fall. « Grapevine Virology in the Third-Generation Sequencing Era : From Virus Detection to Viral Epitranscriptomics ». Plants 10, no 11 (31 octobre 2021) : 2355. http://dx.doi.org/10.3390/plants10112355.
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Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus–plant interactions, as well as the characterization of viral RNA modifications.
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Soltani, Nourolah, Kristian A. Stevens, Vicki Klaassen, Min-Sook Hwang, Deborah A. Golino et Maher Al Rwahnih. « Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics ». Viruses 13, no 6 (11 juin 2021) : 1130. http://dx.doi.org/10.3390/v13061130.
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Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.
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Nellimarla, Srinivas, et Prasad Kesanakurti. « Next-Generation Sequencing : A Promising Tool for Vaccines and Other Biological Products ». Vaccines 11, no 3 (23 février 2023) : 527. http://dx.doi.org/10.3390/vaccines11030527.
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Next-generation sequencing (NGS), also known as high-throughput sequencing (HTS), is a commonly used term to represent a set of DNA sequencing technologies that have been in use for almost two decades [...]
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Wu, David, Ryan O. Emerson, Anna Sherwood, Mignon L. Loh, Anne Angiolillo, Ilan Kirsch, Christopher S. Carlson, David Williamson, Brent L. Wood et Harlan Robins. « Robust Detection Of Minimal Residual Disease In Unselected Patients With B-Cell Precursor Acute Lymphoblastic Leukemia By High-Throughput Sequencing Of IGH ». Blood 122, no 21 (15 novembre 2013) : 2550. http://dx.doi.org/10.1182/blood.v122.21.2550.2550.
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Abstract High-throughput sequencing (HTS) of immunoglobulin heavy chain genes (IGH) may be useful for detecting minimal residual disease (MRD) in acute lymphoblastic leukemia. We previously demonstrated the first application of high-throughput sequencing for the detection of minimal residual disease in T-cell precursor acute lymphoblastic leukemia (TPC-ALL) (Sci. Transl. Med. 4(134):134ra63. 2012). Recently, Faham and colleagues considered deep sequencing for MRD detection in B-cell precursor acute lymphoblastic leukemia (BPC-ALL) (Blood 120(26):5173-80, 2012). As this prior analysis in BPC-ALL apparently focused only on samples known to have a clonal rearrangement in IGH, the potential applicability and wide-spread utility of sequencing of IGH in unselected clinical samples for MRD has not been tested. Here, we consider an unselected cohort of patients enrolled in Children Oncology Group AALL0932 trial and use residual material from 99 patient samples submitted for routine multi-parametric flow cytometry (mpFC) at U. of Washington. One sample failed in the initial DNA extraction step and was not further considered. We show using high-throughput sequencing that clonal IGH rearrangements can be identified in 92 of the remaining 98 pre-treatment samples, using a definition of a V-D-J or D-J rearrangement comprising at least 10% of total nucleated cells (Fig. 1A). Similar to our prior findings in TPC-ALL, we find three subsets of patients—1) those for whom MRD is not detected by either flow cytometry or HTS; 2) those for whom MRD is detected both by flow cytometry and HTS; and 3) those for whom MRD is detected only by HTS, but not flow cytometry (Fig. 1B). There were no false negative results by HTS as compared to flow cytometry.Figure 1Measurement of clonal IGH rearrangement by high-throughput sequencing (HTS) or immunphenotypically abnormal B lymphoblast population by multi-parametric flow cytometry in pre-treatment (A) or day 29 post-treatment (B) residual samples. Results are reported for both HTS (red) and mpFC (blue) as clone frequency per total nucleated cells.Figure 1. Measurement of clonal IGH rearrangement by high-throughput sequencing (HTS) or immunphenotypically abnormal B lymphoblast population by multi-parametric flow cytometry in pre-treatment (A) or day 29 post-treatment (B) residual samples. Results are reported for both HTS (red) and mpFC (blue) as clone frequency per total nucleated cells. In the third group (HTS+positive, flow cytometry-negative), a subset of these patients, (5 of 28) had MRD detectable by HTS at a level within the expected sensitivity of flow cytometry. We hypothesized that in these cases that post-treatment MRD sequences may be present within the maturing B cell compartment that is not immunophenotypically aberrant by flow cytometry. To test this hypothesis, we analyzed eight additional post-treatment samples that were negative for MRD by flow cytometry. The mature B-cell fraction was collected by triple, flow cytometry-sorting and then sequenced by HTS for IGH rearrangements to search for the index clone defined in the corresponding, paired pre-treatment samples. Although a limited finding, diagnostic index IGH sequence was indeed identified in one of eight samples, in only the mature B-cell fraction, which is consistent with the proportion of cases with high-level MRD detected by HTS but which was missed by flow cytometry. Taken together, our results provide additional support for assessment of MRD in acute lymphoblastic leukemia by high-throughput sequencing. Our findings argue that precise quantification of the level of MRD by HTS will be important, and suggest that clonal IGH rearrangement sequences may be detected in an immunophenotypically normal population of mature B cells that may not be detected by flow cytometry. Disclosures: Emerson: Adaptive Biotechnologies: Employment, Equity Ownership. Sherwood:Adaptive Biotechnologies: Employment, Equity Ownership. Kirsch:Adaptive Biotechnologies: Employment, Equity Ownership. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Williamson:Adaptive Biotechnologies: Employment, Equity Ownership. Wood:Becton Dickinson and Company, NJ, USA: Research Funding. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.
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Bai, Ling, Liu He, Penghao Yu, Jiaoyang Luo, Meihua Yang, Xiangren A et Xiaoxing Wei. « Molecular Characterization of Mycobiota and Aspergillus Species from Eupolyphaga sinensis Walker Based on High-Throughput Sequencing of ITS1 and CaM ». Journal of Food Quality 2020 (7 mai 2020) : 1–7. http://dx.doi.org/10.1155/2020/1752415.
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Eupolyphaga sinensis Walker is a valuable traditional Chinese animal medicine first recorded in Shennong Bencao. Previous research has shown that E. sinensis is easily contaminated by aflatoxins (AFs), which are highly toxic mycotoxins, during harvest, storage, and transport, thereby posing a considerable threat to consumer health. Most often, these AFs are produced by Aspergillus species. In this study, we contrast the traditional culture-based dilution plating method to the high-throughput sequencing (HTS) technology for fungal identification in TCM E. sinensis. Both of the methods used internal transcribed spacer 1 (ITS1) and calmodulin (CaM) sequencing for fungal molecular identification. The new CaM primer we designed in the study is suitable for MiSeq PE300 sequencing used for identification of Aspergillus species in community DNA samples. More fungal species were found in the E. sinensis samples based on HTS than those found using the culture-based dilution plating method. Overall, combining the sequencing power of ITS1 and CaM is an effective method for the detection and monitoring of potential toxigenic Aspergillus species in E. sinensis. In conclusion, HTS can be used to obtain a large amount of sequencing data about fungi contaminating animal medicine, allowing earlier detection of potential toxigenic fungi and ensuring the efficient production and safety of E. sinensis.
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Espindola, Andres S., et Kitty F. Cardwell. « Microbe Finder (MiFi®) : Implementation of an Interactive Pathogen Detection Tool in Metagenomic Sequence Data ». Plants 10, no 2 (28 janvier 2021) : 250. http://dx.doi.org/10.3390/plants10020250.
Texte intégralRésumé :
Agricultural high throughput diagnostics need to be fast, accurate and have multiplexing capacity. Metagenomic sequencing is being widely evaluated for plant and animal diagnostics. Bioinformatic analysis of metagenomic sequence data has been a bottleneck for diagnostic analysis due to the size of the data files. Most available tools for analyzing high-throughput sequencing (HTS) data require that the user have computer coding skills and access to high-performance computing. To overcome constraints to most sequencing-based diagnostic pipelines today, we have developed Microbe Finder (MiFi®). MiFi® is a web application for quick detection and identification of known pathogen species/strains in raw, unassembled HTS metagenomic data. HTS-based diagnostic tools developed through MiFi® must pass rigorous validation, which is outlined in this manuscript. MiFi® allows researchers to collaborate in the development and validation of HTS-based diagnostic assays using MiProbe™, a platform used for developing pathogen-specific e-probes. Validated e-probes are made available to diagnosticians through MiDetect™. Here we describe the e-probe development, curation and validation process of MiFi® using grapevine pathogens as a model system. MiFi® can be used with any pathosystem and HTS platform after e-probes have been validated.
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Ruiz-García, Ana Belén, Celia Canales, Félix Morán, Manuel Ruiz-Torres, Magdalena Herrera-Mármol et Antonio Olmos. « Characterization of Spanish Olive Virome by High Throughput Sequencing Opens New Insights and Uncertainties ». Viruses 13, no 11 (6 novembre 2021) : 2233. http://dx.doi.org/10.3390/v13112233.
Texte intégralRésumé :
The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.
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Scherer, Florian, David M. Kurtz, Maximilian Diehn et Ash A. Alizadeh. « High-throughput sequencing for noninvasive disease detection in hematologic malignancies ». Blood 130, no 4 (27 juillet 2017) : 440–52. http://dx.doi.org/10.1182/blood-2017-03-735639.
Texte intégralRésumé :
Abstract Noninvasive monitoring of minimal residual disease (MRD) has led to significant advances in personalized management of patients with hematologic malignancies. Improved therapeutic options and prolonged survival have further increased the need for sensitive tumor assessment that can inform treatment decisions and patient outcomes. At diagnosis or relapse of most hematologic neoplasms, malignant cells are often easily accessible in the blood as circulating tumor cells (CTCs), making them ideal targets to noninvasively profile the molecular features of each patient. In other cancer types, CTCs are generally rare and noninvasive molecular detection relies on circulating tumor DNA (ctDNA) shed from tumor deposits into circulation. The ability to precisely detect and quantify CTCs and ctDNA could minimize invasive procedures and improve prediction of clinical outcomes. Technical advances in MRD detection methods in recent years have led to reduced costs and increased sensitivity, specificity, and applicability. Among currently available tests, high-throughput sequencing (HTS)–based approaches are increasingly attractive for noninvasive molecular testing. HTS-based methods can simultaneously identify multiple genetic markers with high sensitivity and specificity without individual optimization. In this review, we present an overview of techniques used for noninvasive molecular disease detection in selected myeloid and lymphoid neoplasms, with a focus on the current and future role of HTS-based assays.
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Bérubé, Jean A., Patrick N. Gagné, Julien P. Ponchart, J. Phelan, A. Varga et D. James. « Heterobasidion species detected using High Throughput Sequencing (HTS) methods on British Columbia nursery plants ». Canadian Journal of Plant Pathology 41, no 4 (14 juin 2019) : 560–65. http://dx.doi.org/10.1080/07060661.2019.1611665.
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Krehenwinkel, Pomerantz et Prost. « Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies : Current Uses and Future Directions ». Genes 10, no 11 (29 octobre 2019) : 858. http://dx.doi.org/10.3390/genes10110858.
Texte intégralRésumé :
We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology’s MinION portable sequencing platform, as well as summarize areas of recent development.
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Bastida, José María, Rocío Benito, María Luisa Lozano, Ana Marín-Quilez, Kamila Janusz, Marta Martín-Izquierdo, Jesús Hernández-Sánchez et al. « Molecular Diagnosis of Inherited Coagulation and Bleeding Disorders ». Seminars in Thrombosis and Hemostasis 45, no 07 (30 avril 2019) : 695–707. http://dx.doi.org/10.1055/s-0039-1687889.
Texte intégralRésumé :
AbstractDiagnosis of inherited bleeding disorders (IBDs) remains challenging, especially in the case of inherited platelet disorders, due to the heterogeneity of the clinical and laboratory phenotype, the limited specificity of platelet function tests, and the large number of potential culprit genes. Unraveling the underlying molecular defect provides the definitive diagnosis of IBDs, facilitating prognosis and clinical care, which are especially important for severe clinical syndromes and those that may be associated with an increased risk of malignancy. Until recently, Sanger sequencing of candidate genes has been the only method of molecular diagnosis, but this approach is time-consuming and costly and requires phenotype-based identification of any obvious candidate gene(s). Nowadays, high-throughput sequencing (HTS) allows the simultaneous and rapid investigation of multiple genes at a manageable cost. This HTS technology that includes targeted sequencing of prespecified genes, whole-exome sequencing, or whole-genome sequencing, is revolutionizing the genetic diagnosis of human diseases. Through its extensive implementation in research and clinical practice, HTS is rapidly improving the molecular characterization of IBDs. However, despite the availability of this powerful approach, many patients still do not receive a diagnosis. As IBDs are complex and rare diseases, development of more advanced laboratory assays, improvements in bioinformatic pipelines, and the formation of multidisciplinary teams are encouraged to advance our understanding of IBDs.
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Yin, Mengxue, et Wenxing Xu. « Special Issue : “Evolution, Ecology and Diversity of Plant Virus” ». Viruses 15, no 2 (9 février 2023) : 487. http://dx.doi.org/10.3390/v15020487.
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Choi, Jiyeong, Anya Clara Osatuke, Griffin Erich, Kristian Stevens, Min Sook Hwang, Maher Al Rwahnih et Marc Fuchs. « High-Throughput Sequencing Reveals Tobacco and Tomato Ringspot Viruses in Pawpaw ». Plants 11, no 24 (17 décembre 2022) : 3565. http://dx.doi.org/10.3390/plants11243565.
Texte intégralRésumé :
Pawpaw (Asimina triloba) trees exhibiting stunting and foliar mosaic, chlorosis, or distortions were observed in New York. In 2021, leaf samples from two symptomatic trees and a sapling, as well as two asymptomatic trees, were tested for the presence of viruses and viroids by high-throughput sequencing (HTS) using total RNA after ribosomal RNA depletion. HTS sequence information revealed tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) in symptomatic but not in asymptomatic leaves. HTS reads and de novo-assembled contigs covering the genomes of both viruses were obtained, with a higher average read depth for RNA2 than RNA1. The occurrence of TRSV and ToRSV was confirmed in the original leaf samples used for HTS and 12 additional trees and saplings from New York and Maryland in 2022 by RT-PCR combined with Sanger sequencing, and DAS-ELISA. Single infections by TRSV in 11 of 14 trees and dual infections by TRSV and ToRSV in 3 of 14 trees were identified. The nucleotide sequence identity of partial gene fragments of TRSV and ToRSV was high among pawpaw isolates (94.9–100% and 91.8–100%, respectively) and between pawpaw isolates and isolates from other horticultural crops (93.6–100% and 71.3–99.3%, respectively). This study is the first to determine the virome of pawpaw.
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Kutnjak, Denis, Lucie Tamisier, Ian Adams, Neil Boonham, Thierry Candresse, Michela Chiumenti, Kris De Jonghe et al. « A Primer on the Analysis of High-Throughput Sequencing Data for Detection of Plant Viruses ». Microorganisms 9, no 4 (14 avril 2021) : 841. http://dx.doi.org/10.3390/microorganisms9040841.
Texte intégralRésumé :
High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.
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Lightbody, Gaye, Valeriia Haberland, Fiona Browne, Laura Taggart, Huiru Zheng, Eileen Parkes et Jaine K. Blayney. « Review of applications of high-throughput sequencing in personalized medicine : barriers and facilitators of future progress in research and clinical application ». Briefings in Bioinformatics 20, no 5 (14 juin 2019) : 1795–811. http://dx.doi.org/10.1093/bib/bby051.
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Abstract There has been an exponential growth in the performance and output of sequencing technologies (omics data) with full genome sequencing now producing gigabases of reads on a daily basis. These data may hold the promise of personalized medicine, leading to routinely available sequencing tests that can guide patient treatment decisions. In the era of high-throughput sequencing (HTS), computational considerations, data governance and clinical translation are the greatest rate-limiting steps. To ensure that the analysis, management and interpretation of such extensive omics data is exploited to its full potential, key factors, including sample sourcing, technology selection and computational expertise and resources, need to be considered, leading to an integrated set of high-performance tools and systems. This article provides an up-to-date overview of the evolution of HTS and the accompanying tools, infrastructure and data management approaches that are emerging in this space, which, if used within in a multidisciplinary context, may ultimately facilitate the development of personalized medicine.
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Fabiańska, Izabela, Stefan Borutzki, Benjamin Richter, Hon Q. Tran, Andreas Neubert et Dietmar Mayer. « LABRADOR—A Computational Workflow for Virus Detection in High-Throughput Sequencing Data ». Viruses 13, no 12 (18 décembre 2021) : 2541. http://dx.doi.org/10.3390/v13122541.
Texte intégralRésumé :
High-throughput sequencing (HTS) allows detection of known and unknown viruses in samples of broad origin. This makes HTS a perfect technology to determine whether or not the biological products, such as vaccines are free from the adventitious agents, which could support or replace extensive testing using various in vitro and in vivo assays. Due to bioinformatics complexities, there is a need for standardized and reliable methods to manage HTS generated data in this field. Thus, we developed LABRADOR—an analysis pipeline for adventitious virus detection. The pipeline consists of several third-party programs and is divided into two major parts: (i) direct reads classification based on the comparison of characteristic profiles between reads and sequences deposited in the database supported with alignment of to the best matching reference sequence and (ii) de novo assembly of contigs and their classification on nucleotide and amino acid levels. To meet the requirements published in guidelines for biologicals’ safety we generated a custom nucleotide database with viral sequences. We tested our pipeline on publicly available HTS datasets and showed that LABRADOR can reliably detect viruses in mixtures of model viruses, vaccines and clinical samples.
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Giner, Caterina R., Irene Forn, Sarah Romac, Ramiro Logares, Colomban de Vargas et Ramon Massana. « Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes ». Applied and Environmental Microbiology 82, no 15 (27 mai 2016) : 4757–66. http://dx.doi.org/10.1128/aem.00560-16.
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ABSTRACTHigh-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescencein situhybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances.IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescencein situhybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed.
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Grardel, Nathalie, Mikaël Salson, Aurélie Caillault, Marc Duez, Céline Villenet, Christophe Roumier, Martin Figeac et al. « Multiclonal Diagnosis and MRD Follow-up in ALL with HTS Coupled with a Bioinformatic Analysis ». Blood 124, no 21 (6 décembre 2014) : 1083. http://dx.doi.org/10.1182/blood.v124.21.1083.1083.
Texte intégralRésumé :
Abstract Background: The molecular diagnosis in ALL allows by the research for rearrangements V(D)J on lymphoblast DNA , to find markers of clonality in 95% of the cases. These markers also are used to quantify the minimal residual disease by real time Q-PCR to adapt treatments. This strategy fails in some cases : Absence of initial marker, failure of sequencing or emergence at relapse time of a clone not observed at diagnosis time or in very minority. Several studies have asserted the usefulness of high-throughput sequencing (HTS). It enables deep sequencing of a lymphoid population, bypassing some of these problems. However, the huge amount of data raises two challenges. First, hospitals must be able to store and process terabytes of data per year. Second, the data must be nicely synthesized to ease clinician interpretation. Here, we report the use of HTS, in a hematology lab, for diagnosis and follow-up of ALL combined with a bioinformatic analysis and visualization with the new dedicated Vidjil software (Giraud, Salson, et al, BMC Genomics 2014, http://www.vidjil.org). Patients and methods: We studied the clonality of 8 pediatric patients (5 B-ALL and 3 T-ALL, 2w/6m, 2-14 years) at diagnosis and follow-up (37 follow-up time points). The sensitivity was estimated by a range of dilution of DNA tumoral in DNA of PBL from healthy donors (10-2 to 10-5). For every sample, 500ng of bone marrow DNA are extracted on Qiagen® Kit, measured on NanoDrop system® and amplified by a classical (not fluorescent) PCR system for TCRg and IgH target. These systems are described or derived from the BIOMED-2 works. The sequencing libraries are done from the PCR products, verified by electrophoresis on agarose gel then bar-coded with Ion Fragment Plus® kit and sequenced with an Ion Torrent® 318 Chip system. The obtained sequences are classified on the basis of their V(D)J rearrangements. The dedicated Vidjil browser enables to explore the lymphocyte population and to track the clones along the time. We can inspect the sequences and send them directly to IMGT/V-QUEST or IgBlast for further analysis. It is possible to tag, rename or filter out some clones, and export the resulting graphs to a printable file. Due to sequencing errors, there may be several clones corresponding to a real clone. The browser enables to align such sequences, and we can choose to merge them. The browser can also be used to compare several runs on the same sample, for example with different PCR conditions. Results: We identified several clones in the diagnosis sample and observed their evolution at different follow-up time points. Clones that were detected by classic methods were also found by Vidjil. Moreover the software allows us to look more in-depth at other clones appearing at lower concentrations. Relapses were detected, and for one patient, two emerging clones were observed. Figure 1 shows plots of the concentration for a patient with B common-ALL. The first point is the diagnosis; the four other points are respectively D35, D70, and D90 after bone marrow transplantation, and relapse. The patient was followed both on IgH (upper plot) and TCRg (lower plot). In both systems, there is the emergence of a new clone at relapse while some of the main clones at diagnosis were not detectable anymore or at a very low concentration. Those clones were also confirmed by conventional methods. Figure 1 Figure 1. Using high-throughput sequencing together with bioinformatic analysis and visualization with Vidjil allows identifying very easily the emergence of new clones that were not detected at diagnosis. Conclusion: The HTS prefigures new steps both for the knowledge of the lymphoid and auto-immune pathologies and for the ALL MRD follow-up. Coupled with a bioinformatic analysis, it gives a more complete insight of the blastic population at diagnosis and allows observing the evolution of this population. The whole analysis including the preparation, the sequencing, the software analysis and the clinician validation seems faster than the current protocols. Our protocol has been designed and tested for two years in Lille and is now being tested in other French hospitals involved in ALL-MRD. We believe that such integrated approaches, where clinicians maintain control over patient data, have their role to play. This raises the need for people having experience with high-throughput sequencing in hospitals. With the advent of this technology and its biomedical applications, that should not be a great issue. Disclosures No relevant conflicts of interest to declare.
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Lynch, Tarah, Aaron Petkau, Natalie Knox, Morag Graham et Gary Van Domselaar. « A Primer on Infectious Disease Bacterial Genomics ». Clinical Microbiology Reviews 29, no 4 (7 septembre 2016) : 881–913. http://dx.doi.org/10.1128/cmr.00001-16.
Texte intégralRésumé :
SUMMARYThe number of large-scale genomics projects is increasing due to the availability of affordable high-throughput sequencing (HTS) technologies. The use of HTS for bacterial infectious disease research is attractive because one whole-genome sequencing (WGS) run can replace multiple assays for bacterial typing, molecular epidemiology investigations, and more in-depth pathogenomic studies. The computational resources and bioinformatics expertise required to accommodate and analyze the large amounts of data pose new challenges for researchers embarking on genomics projects for the first time. Here, we present a comprehensive overview of a bacterial genomics projects from beginning to end, with a particular focus on the planning and computational requirements for HTS data, and provide a general understanding of the analytical concepts to develop a workflow that will meet the objectives and goals of HTS projects.
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Mu, Yawen, Chao Song, Jianghua Yang, Yong Zhang et Xiaowei Zhang. « Next-Generation DNA Barcoding for Fish Identification Using High-Throughput Sequencing in Tai Lake, China ». Water 15, no 4 (16 février 2023) : 774. http://dx.doi.org/10.3390/w15040774.
Texte intégralRésumé :
Tai Lake, an important biodiversity hotspot of the lower reaches of the Yangtze River in China, possesses its own characteristic fish fauna. Barcoding on native species is important for species identification and biodiversity assessment with the emerged molecular-based method, such as environmental DNA (eDNA) metabarcoding. Here, DNA-barcoding coupled with high-throughput sequencing (HTS) and traditional Sanger sequencing were introduced to barcoding 180 specimens belonging to 33 prior morphological species, including the most majority of fish fauna in Tai Lake. HTS technology, on the one hand, significant enhances the capture of barcode sequences of fish. The successful rate of fish barcoding was 74% and 91% in Sanger and HTS, respectively. On the other hand, the HTS output has a large number (64%) of insertions and deletions, which require strict bioinformatics processing to ensure that the ‘‘true’’ barcode sequence is captured. Cross-contamination and parasites were the main error sources that compromised attempts at the DNA barcoding of fish species. The barcode gap analysis was 100% successful at delimiting species in all specimens. The automatic barcode gap discovery (ABGD) method grouped barcode sequences into 34 OTUs, and some deep divergence and closed species failed to obtain corresponding OTUs. Overall, the local species barcode library established by HTS barcoding here is anticipated to shed a new light on the conservation of fish diversity in Tai Lake.
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Minicka, Julia, Aleksandra Zarzyńska-Nowak, Daria Budzyńska, Natasza Borodynko-Filas et Beata Hasiów-Jaroszewska. « High-Throughput Sequencing Facilitates Discovery of New Plant Viruses in Poland ». Plants 9, no 7 (29 juin 2020) : 820. http://dx.doi.org/10.3390/plants9070820.
Texte intégralRésumé :
Viruses cause epidemics on all major crops of agronomic importance, and a timely and accurate identification is essential for control. High throughput sequencing (HTS) is a technology that allows the identification of all viruses without prior knowledge on the targeted pathogens. In this paper, we used HTS technique for the detection and identification of different viral species occurring in single and mixed infections in plants in Poland. We analysed various host plants representing different families. Within the 20 tested samples, we identified a total of 13 different virus species, including those whose presence has not been reported in Poland before: clover yellow mosaic virus (ClYMV) and melandrium yellow fleck virus (MYFV). Due to this new finding, the obtained sequences were compared with others retrieved from GenBank. In addition, cucurbit aphid-borne yellows virus (CABYV) was also detected, and due to the recent occurrence of this virus in Poland, a phylogenetic analysis of these new isolates was performed. The analysis revealed that CABYV population is highly diverse and the Polish isolates of CABYV belong to two different phylogenetic groups. Our results showed that HTS-based technology is a valuable diagnostic tool for the identification of different virus species originating from variable hosts, and can provide rapid information about the spectrum of plant viruses previously not detected in a region.
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Vasselon, Valentin, Isabelle Domaizon, Frédéric Rimet, Maria Kahlert et Agnès Bouchez. « Application of high-throughput sequencing (HTS) metabarcoding to diatom biomonitoring : Do DNA extraction methods matter ? » Freshwater Science 36, no 1 (mars 2017) : 162–77. http://dx.doi.org/10.1086/690649.
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Zhou, Sisi, Yonggui Fu, Jie Li, Lingyu He, Xingsheng Cai, Qingyu Yan, Xingqiang Rao et al. « HTS-PEG : A Method for High Throughput Sequencing of the Paired-Ends of Genomic Libraries ». PLoS ONE 7, no 12 (20 décembre 2012) : e52257. http://dx.doi.org/10.1371/journal.pone.0052257.
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Bokulich, Nicholas A., et David A. Mills. « Improved Selection of Internal Transcribed Spacer-Specific Primers Enables Quantitative, Ultra-High-Throughput Profiling of Fungal Communities ». Applied and Environmental Microbiology 79, no 8 (1 février 2013) : 2519–26. http://dx.doi.org/10.1128/aem.03870-12.
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ABSTRACTUltra-high-throughput sequencing (HTS) of fungal communities has been restricted by short read lengths and primer amplification bias, slowing the adoption of newer sequencing technologies to fungal community profiling. To address these issues, we evaluated the performance of several common internal transcribed spacer (ITS) primers and designed a novel primer set and work flow for simultaneous quantification and species-level interrogation of fungal consortia. Primer comparison and validation were predictedin silicoand by sequencing a “mock community” of mixed yeast species to explore the challenges of amplicon length and amplification bias for reconstructing defined yeast community structures. The amplicon size and distribution of this primer set are smaller than for all preexisting ITS primer sets, maximizing sequencing coverage of hypervariable ITS domains by very-short-amplicon, high-throughput sequencing platforms. This feature also enables the optional integration of quantitative PCR (qPCR) directly into the HTS preparatory work flow by substituting qPCR with these primers for standard PCR, yielding quantification of individual community members. The complete work flow described here, utilizing any of the qualified primer sets evaluated, can rapidly profile mixed fungal communities and capably reconstructed well-characterized beer and wine fermentation fungal communities.
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Zhuang, Fanglei, Ryan T. Fuchs et G. Brett Robb. « Small RNA Expression Profiling by High-Throughput Sequencing : Implications of Enzymatic Manipulation ». Journal of Nucleic Acids 2012 (2012) : 1–15. http://dx.doi.org/10.1155/2012/360358.
Texte intégralRésumé :
Eukaryotic regulatory small RNAs (sRNAs) play significant roles in many fundamental cellular processes. As such, they have emerged as useful biomarkers for diseases and cell differentiation states. sRNA-based biomarkers outperform traditional messenger RNA-based biomarkers by testing fewer targets with greater accuracy and providing earlier detection for disease states. Therefore, expression profiling of sRNAs is fundamentally important to further advance the understanding of biological processes, as well as diagnosis and treatment of diseases. High-throughput sequencing (HTS) is a powerful approach for both sRNA discovery and expression profiling. Here, we discuss the general considerations for sRNA-based HTS profiling methods from RNA preparation to sequencing library construction, with a focus on the causes of systematic error. By examining the enzymatic manipulation steps of sRNA expression profiling, this paper aims to demystify current HTS-based sRNA profiling approaches and to aid researchers in the informed design and interpretation of profiling experiments.
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Lambert, Christophe, Cassandra Braxton, Robert Charlebois, Avisek Deyati, Paul Duncan, Fabio La Neve, Heather Malicki et al. « Considerations for Optimization of High-Throughput Sequencing Bioinformatics Pipelines for Virus Detection ». Viruses 10, no 10 (27 septembre 2018) : 528. http://dx.doi.org/10.3390/v10100528.
Texte intégralRésumé :
High-throughput sequencing (HTS) has demonstrated capabilities for broad virus detection based upon discovery of known and novel viruses in a variety of samples, including clinical, environmental, and biological. An important goal for HTS applications in biologics is to establish parameter settings that can afford adequate sensitivity at an acceptable computational cost (computation time, computer memory, storage, expense or/and efficiency), at critical steps in the bioinformatics pipeline, including initial data quality assessment, trimming/cleaning, and assembly (to reduce data volume and increase likelihood of appropriate sequence identification). Additionally, the quality and reliability of the results depend on the availability of a complete and curated viral database for obtaining accurate results; selection of sequence alignment programs and their configuration, that retains specificity for broad virus detection with reduced false-positive signals; removal of host sequences without loss of endogenous viral sequences of interest; and use of a meaningful reporting format, which can retain critical information of the analysis for presentation of readily interpretable data and actionable results. Furthermore, after alignment, both automated and manual evaluation may be needed to verify the results and help assign a potential risk level to residual, unmapped reads. We hope that the collective considerations discussed in this paper aid toward optimization of data analysis pipelines for virus detection by HTS.
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Ercolini, Danilo. « High-Throughput Sequencing and Metagenomics : Moving Forward in the Culture-Independent Analysis of Food Microbial Ecology ». Applied and Environmental Microbiology 79, no 10 (8 mars 2013) : 3148–55. http://dx.doi.org/10.1128/aem.00256-13.
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ABSTRACTFollowing recent trends in environmental microbiology, food microbiology has benefited from the advances in molecular biology and adopted novel strategies to detect, identify, and monitor microbes in food. An in-depth study of the microbial diversity in food can now be achieved by using high-throughput sequencing (HTS) approaches after direct nucleic acid extraction from the sample to be studied. In this review, the workflow of applying culture-independent HTS to food matrices is described. The current scenario and future perspectives of HTS uses to study food microbiota are presented, and the decision-making process leading to the best choice of working conditions to fulfill the specific needs of food research is described.
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Rea, Bryan, Paul Haun, Ryan Emerson, Marissa Vignali, Midhat Farooqi, Sara Samimi, Rosalie Elenitsas, Ilan Kirsch et Adam Bagg. « Role of high-throughput sequencing in the diagnosis of cutaneous T-cell lymphoma ». Journal of Clinical Pathology 71, no 9 (10 avril 2018) : 814–20. http://dx.doi.org/10.1136/jclinpath-2018-205004.
Texte intégralRésumé :
AimsSubstantial clinicopathological overlap exists between cutaneous T-cell lymphoma (CTCL) and benign conditions, leading to diagnostic difficulties. We sought to delineate the utility of high-throughput sequencing (HTS) across a spectrum of histological findings in CTCL and reactive mimics.MethodsOne hundred skin biopsies obtained for clinical concern for CTCL were identified, comprising 25 cases each from four histological categories: ‘definitive CTCL’, ‘atypical lymphoid infiltrate, concerning for CTCL’, ‘atypical lymphoid infiltrate, favour reactive’ or ‘reactive lymphoid infiltrate’. T-cell receptor gamma chain gene (TRG) PCR and T-cell receptor beta chain gene HTS were performed on both skin biopsy and concurrently collected peripheral blood; most peripheral blood samples were also analysed by flow cytometry.ResultsHistologically defined CTCL specimens had significantly higher clonality scores and T-cell fractions via HTS than all other groups (all p<0.002 and p<0.03, respectively). HTS was more diagnostically specific than TRG PCR in skin (100% vs 88%), while diagnostic sensitivity (68% vs 72%) and accuracy (84% vs 80%) were similar. TRG PCR and flow cytometry performed on blood were the least diagnostically useful assays. Some identically sized peaks detected by TRG PCR in concurrent skin and peripheral blood specimens were non-identical by HTS analysis.ConclusionsHTS, by assessing both clonality and T-cell fractions in skin biopsies, is a powerful tool to aid in the diagnosis of CTCL. It is more specific than TRG PCR in distinguishing definitive CTCL from reactive and indeterminate histology. Identically sized peaks by TRG PCR, typically interpreted to be clonally related, are not always clonally identical by sequencing.
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Blanco, Celia, Samuel Verbanic, Burckhard Seelig et Irene A. Chen. « High throughput sequencing of in vitro selections of mRNA-displayed peptides : data analysis and applications ». Physical Chemistry Chemical Physics 22, no 12 (2020) : 6492–506. http://dx.doi.org/10.1039/c9cp05912a.
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Ng, Siemon, Cassandra Braxton, Marc Eloit, Szi Feng, Romain Fragnoud, Laurent Mallet, Edward Mee, Sarmitha Sathiamoorthy, Olivier Vandeputte et Arifa Khan. « Current Perspectives on High-Throughput Sequencing (HTS) for Adventitious Virus Detection : Upstream Sample Processing and Library Preparation ». Viruses 10, no 10 (16 octobre 2018) : 566. http://dx.doi.org/10.3390/v10100566.
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A key step for broad viral detection using high-throughput sequencing (HTS) is optimizing the sample preparation strategy for extracting viral-specific nucleic acids since viral genomes are diverse: They can be single-stranded or double-stranded RNA or DNA, and can vary from a few thousand bases to over millions of bases, which might introduce biases during nucleic acid extraction. In addition, viral particles can be enveloped or non-enveloped with variable resistance to pre-treatment, which may influence their susceptibility to extraction procedures. Since the identity of the potential adventitious agents is unknown prior to their detection, efficient sample preparation should be unbiased toward all different viral types in order to maximize the probability of detecting any potential adventitious viruses using HTS. Furthermore, the quality assessment of each step for sample processing is also a critical but challenging aspect. This paper presents our current perspectives for optimizing upstream sample processing and library preparation as part of the discussion in the Advanced Virus Detection Technologies Interest group (AVDTIG). The topics include: Use of nuclease treatment to enrich for encapsidated nucleic acids, techniques for amplifying low amounts of virus nucleic acids, selection of different extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation.
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Wood, Brent, David Wu, Beryl Crossley, Yunfeng Dai, David Williamson, Charles Gawad, Michael J. Borowitz et al. « Measurable residual disease detection by high-throughput sequencing improves risk stratification for pediatric B-ALL ». Blood 131, no 12 (22 mars 2018) : 1350–59. http://dx.doi.org/10.1182/blood-2017-09-806521.
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Key Points HTS identifies MRD at the conventional clinical cutoff in more patients than FC, and these patients have worse outcomes. A subset of B-ALL patients essentially cured using current chemotherapy is identified at end of induction by HTS.
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Espindola, Andres S., Daniela Sempertegui-Bayas, Danny F. Bravo-Padilla, Viviana Freire-Zapata, Francisco Ochoa-Corona et Kitty F. Cardwell. « TASPERT : Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics ». Viruses 13, no 7 (24 juin 2021) : 1223. http://dx.doi.org/10.3390/v13071223.
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High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique’s success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.