scholarly journals Assessment of Antibody Library Diversity through Next Generation Sequencing and Technical Error Compensation.

2016 ◽  
Author(s):  
Marco Fantini ◽  
Luca Pandolfini ◽  
Simonetta Lisi ◽  
Michele Chirichella ◽  
Ivan Arisi ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Small Sample ◽  
Sequencing Error ◽  
Limited Information ◽  
Next Generation ◽  
Antibody Library ◽  
Illumina Platform ◽  
Wide Range ◽  
Antibody Libraries ◽  
Generation Sequencing

Antibody libraries are important resources to derive antibodies to be used for a wide range of applications, from structural and functional studies to intracellular protein interference studies to developing new diagnostics and therapeutics. Whatever the goal, the key parameter for an antibody library is its diversity, i.e. the number of distinct elements in the collection, which directly reflects the probability of finding in the library an antibody against a given antigen, of sufficiently high affinity. Quantitative evaluation of antibody library diversity and quality has been for a long time inadequately addressed, due to the high similarity and length of the sequences of the library. Diversity was usually inferred by the transformation efficiency and tested either by fingerprinting and/or sequencing of a few hundred random library elements. Inferring diversity from such a small sample is, however, very rudimental and gives limited information about the real complexity, because complexity does not scale linearly with sample size. Next-generation sequencing (NGS) has opened new ways to tackle the antibody library diversity quality assessment. However, much remains to be done to fully exploit the potential of NGS for the quantitative analysis of antibody repertoires and to overcome current limitations. To obtain a more reliable antibody library complexity estimate here we show a new, PCR-free, NGS approach to sequence antibody libraries on Illumina platform, coupled to a new bioinformatic analysis and software (Diversity Estimator of Antibody Library, DEAL) that allows to reliably estimate the diversity, taking in consideration the sequencing error.

scholarly journals Extracting Small RNAs from Human Biological Fluids for Subsequent Next-Generation Sequencing

2019 ◽  
Vol 9 (1) ◽  
pp. 80-86
Author(s):  
O. A. Beylerli ◽  
A. T Beylerli ◽  
I. F. Garaev
Keyword(s):  
Next Generation Sequencing ◽  
Small Rnas ◽  
Biological Fluids ◽  
Rna Extraction ◽  
Small Sample ◽  
Next Generation ◽  
Illumina Platform ◽  
Small Noncoding Rnas ◽  
Organic Extraction ◽  
Generation Sequencing

A number of questions arise when choosing methods for experiments related to next-generation sequencing. On the one hand, while working with RNA extraction, added reagents and their residues can often inhibit sensitive chemicals with which the sequential synthesis is carried out for the sequencing. On the other hand, processing the same data using different software for the analysis can also impact on the sequencing results. This paper will present the step by step procedure for the preparation of samples taken from human biological fluids for subsequent sequencing of small RNAs, small noncoding RNAs in particular. Regarding the methods of extraction or isolation of RNAs, we found that low RNA yield can be improved significantly by following the isolation method for total RNA and its fractions included in Ambion’s MirVana PARIS kit, but only if using a special approach and modifying the organic extraction step. Compared to others, the methods supplied with commercially available kits at the time of researching this paper require only one organic extraction. This simple but, as it turned out, very useful modification makes it possible to access previously unavailable material. Potential advantages of this modification include a more complete profiling of small non-coding RNAs and a broader access to small sample volumes, as a rule, access to human biological fluids which can be prepared for RNA sequencing on the Illumina platform.

scholarly journals Resolving Differential Diagnostic Problems in von Willebrand Disease, in Fibrinogen Disorders, in Prekallikrein Deficiency and in Hereditary Hemorrhagic Telangiectasia by Next-Generation Sequencing

Life ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 202
Author(s):  
Réka Gindele ◽  
Adrienne Kerényi ◽  
Judit Kállai ◽  
György Pfliegler ◽  
Ágota Schlammadinger ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Hereditary Hemorrhagic Telangiectasia ◽  
Von Willebrand Disease ◽  
Differential Diagnostics ◽  
Bleeding Disorders ◽  
Next Generation ◽  
Illumina Platform ◽  
Clinical Patient ◽  
Von Willebrand ◽  
Generation Sequencing

Diagnosis of rare bleeding disorders is challenging and there are several differential diagnostics issues. Next-generation sequencing (NGS) is a useful tool to overcome these problems. The aim of this study was to demonstrate the usefulness of molecular genetic investigations by summarizing the diagnostic work on cases with certain bleeding disorders. Here we report only those, in whom NGS was indicated due to uncertainty of diagnosis or if genetic confirmation of initial diagnosis was required. Based on clinical and/or laboratory suspicion of von Willebrand disease (vWD, n = 63), hypo-or dysfibrinogenemia (n = 27), hereditary hemorrhagic telangiectasia (HHT, n = 10) and unexplained activated partial thromboplastin time (APTT) prolongation (n = 1), NGS using Illumina platform was performed. Gene panel covered 14 genes (ACVRL1, ENG, MADH4, GDF2, RASA1, F5, F8, FGA, FGB, FGG, KLKB1, ADAMTS13, GP1BA and VWF) selected on the basis of laboratory results. We identified forty-seven mutations, n = 29 (6 novel) in vWD, n = 4 mutations leading to hemophilia A, n = 10 (2 novel) in fibrinogen disorders, n = 2 novel mutations in HHT phenotype and two mutations (1 novel) leading to prekallikrein deficiency. By reporting well-characterized cases using standardized, advanced laboratory methods we add new pieces of data to the continuously developing “bleeding disorders databases”, which are excellent supports for clinical patient management.

scholarly journals Next-Generation Sequencing Identification and Characterization of MicroRNAs in Dwarfed Citrus Trees Infected With Citrus Dwarfing Viroid in High-Density Plantings

2021 ◽  
Vol 12 ◽  
Author(s):  
Tyler Dang ◽  
Irene Lavagi-Craddock ◽  
Sohrab Bodaghi ◽  
Georgios Vidalakis
Keyword(s):  
Next Generation Sequencing ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
High Density ◽  
Poncirus Trifoliata ◽  
Next Generation ◽  
Developmental Processes ◽  
Wide Range ◽  
Generation Sequencing ◽  
Citrus Trees

Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.

scholarly journals Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair derived artifacts as residual errors

2019 ◽  
Author(s):  
Xinyue You ◽  
Suresh Thiruppathi ◽  
Weiying Liu ◽  
Yiyi Cao ◽  
Mikihiko Naito ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Low Frequency ◽  
Repair Process ◽  
Sequencing Error ◽  
Base Substitution ◽  
Next Generation ◽  
Background Error ◽  
Technical Errors ◽  
Genome Wide ◽  
Generation Sequencing

ABSTRACTTo improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strands-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 6 bp of the library fragments in the 5’-NpCpA-3’ or 5’-NpCpT-3’ trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 6 bp, PECC-Seq could reduce the sequencing error frequency to mid-10−7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10−8. In mutagen-treated TK6, slight increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights in further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.

scholarly journals 355. A Novel Likelihood-Based Model to Estimate SARS-CoV-2 Viral Titer from Next-Generation Sequencing Data

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S281-S282
Author(s):  
Heather L Wells ◽  
Joseph Barrows ◽  
Mara Couto-Rodriguez ◽  
Xavier O Jirau Serrano ◽  
Marilyne Debieu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Board Member ◽  
Clinical Specimen ◽  
Next Generation Sequencing Data ◽  
Viral Titer ◽  
Next Generation ◽  
Genome Coverage ◽  
Ct Value ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background The quantitative level of pathogens present in a host is a major driver of infectious disease (ID) state and outcome. However, the majority of ID diagnostics are qualitative. Next-generation sequencing (NGS) is an emerging ID diagnostics and research tool to provide insights, including tracking transmission, evolution, and identifying novel strains. Methods We built a novel likelihood-based computational method to leverage pathogen-specific genome-wide NGS data to detect SARS-CoV-2, profile genetic variants, and furthermore quantify levels of these pathogens. We used de-identified clinical specimens tested for SARS-CoV-2 using RT-PCR, SARS-CoV-2 NGS Assay (hybrid capture, Twist Bioscience), or ARTIC (amplicon-based) platform, and COVID-DX software. A training (n=87) and validation (n=22) set was selected to establish the strength of our quantification model. We fit non-uniform probabilistic error profiles to a deterministic sigmoidal equation that more realistically represents observed data and used likelihood maximized over several different read depths to improve accuracy over a wide range of values of viral load. Given the proportion of the genome covered at varying depths for a single sample as input data, our model estimated the Ct of that sample as the value that produces the maximum likelihood of generating the observed genome coverage data. Results The model fit on 87 SARS-CoV-2 NGS Assay training samples produced a good fit to the 22 validation samples, with a coefficient of correlation (r2) of ~0.8. The accuracy of the model was high (mean absolute % error of ~10%, meaning our model is able to predict the Ct value of each sample within a margin of ±10% on average). Because of the nature of the commonly used ARTIC protocol, we found that all quantitative signals in this data were lost during PCR amplification and the model is not applicable for quantification of samples captured this way. The ability to model quantification is a major advantage of the SARS-CoV-2 NGS assay protocol. The likelihood-based model to estimate SARS-CoV-2 viral titer Left Observed genome coverage (y-axis) plotted against Ct value (x-axis). The best-fitting logistic curve is demonstrated with a red line with shaded areas above and below representing the fitted error profile. RIGHT: Model-estimated Ct values (y-axis) compared to laboratory Ct values (x-axis) with grey bars representing estimated confidence intervals. The 1:1 diagonal is shown as a dotted line. Conclusion To our knowledge, this is the first model to incorporate sequence data mapped across the genome of a pathogen to quantify the level of that pathogen in a clinical specimen. This has implications in ID diagnostics, research, and metagenomics. Disclosures Heather L. Wells, MPH, Biotia, Inc. (Consultant) Joseph Barrows, MS, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Xavier O. Jirau Serrano, B.S., Biotia (Employee) Marilyne Debieu, PhD, Biotia (Employee) Karen Wessel, PhD, Labor Zotz/Klimas (Employee) Christopher Mason, PhD, Biotia (Board Member, Advisor or Review Panel member, Shareholder) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)

scholarly journals 265. Clinical Epidemiology of Invasive Fungal Infection with Aspergillus and Mucor Species in a Tertiary Children’s Hospital

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S147-S147
Author(s):  
Zacharoula Oikonomopoulou ◽  
Sameer Patel ◽  
Jacquie Toia ◽  
William Muller
Keyword(s):  
Next Generation Sequencing ◽  
Fungal Infection ◽  
Invasive Fungal Infection ◽  
Small Sample ◽  
Fungal Culture ◽  
Next Generation ◽  
Hematopoietic Stem ◽  
Children's Hospital ◽  
Children’S Hospital ◽  
Generation Sequencing

Abstract Background Patients undergoing hematopoietic stem cell transplantation and patients with hematologic malignancies are at increased risk for acquiring invasive fungal infection (IFI) due to immune system impairment from chemotherapy. Affected patients require prolonged antifungal therapy with the risk of associated toxicity and extended hospitalization due to delay of accurate diagnosis. There is a lack of effective serologic biomarkers and hesitancy to proceed with tissue diagnosis due to thrombocytopenia or other associated risks. Mortality in oncology patients with invasive mycoses is high, with pediatric mortality rates of 30–40% at 12 weeks following diagnosis. Methods All patients that were admitted to Lurie Children’s Hospital between January 2014 and December 2018 and received voriconazole, ambisome, posaconazole and isavuconazole were identified. The following data were retrospectively collected: CT chest and sinus, (1,3)-β-d-Glucan and Aspergillus galactomannan, ANC and ALC at diagnosis, blood next-generation sequencing, tissue 18s rRNA, fungal culture, duration of neutropenia and lymphopenia, site of infection, time between underlying diagnosis and development of IFI, surgical intervention and associated mortality. Results A total of 94 unique patients that received voriconazole were identified. There were 8 proven cases of invasive Aspergillus infection the past 5 years, 50% male, mean age 14 years. Only 25% of patients had positive serum Aspergillus galactomannan and 37.5% had positive β-d-Glucan. Seven cases were due to Aspergillus fumigatus and one case was due to Aspergillus flavus. There were 9 patients with mucormycosis and all but one were culture positive. Three patients with Mucor had mold identification in blood next-generation sequencing prior to surgery. Mucor associated mortality was 22.2%. Conclusion The majority of pediatric patients with invasive aspergillosis did not have characteristic chest CT imaging findings and serum Aspergillus galactomannan was usually negative.The was no associated mortality in invasive Aspergillus cases, whereas the mortality rate of invasive mucormycosis was 22.2%. Although we have a small sample size, this is significantly lower compared with published literature. Disclosures All authors: No reported disclosures.

Multicenter validation study for the certification of a CFTR gene scanning method using next generation sequencing technology

2018 ◽  
Vol 56 (7) ◽  
pp. 1046-1053 ◽  
Author(s):  
Anne Bergougnoux ◽  
Valeria D’Argenio ◽  
Stefanie Sollfrank ◽  
Fanny Verneau ◽  
Antonella Telese ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Molecular Genetic Analysis ◽  
Analytical Sensitivity ◽  
Next Generation ◽  
Sequencing Data ◽  
Scanning Method ◽  
Next Generation Sequencing Technology ◽  
Wide Range ◽  
Generation Sequencing

Abstract Background: Many European laboratories offer molecular genetic analysis of the CFTR gene using a wide range of methods to identify mutations causative of cystic fibrosis (CF) and CFTR-related disorders (CFTR-RDs). Next-generation sequencing (NGS) strategies are widely used in diagnostic practice, and CE marking is now required for most in vitro diagnostic (IVD) tests in Europe. The aim of this multicenter study, which involved three European laboratories specialized in CF molecular analysis, was to evaluate the performance of Multiplicom’s CFTR MASTR Dx kit to obtain CE-IVD certification. Methods: A total of 164 samples, previously analyzed with well-established “reference” methods for the molecular diagnosis of the CFTR gene, were selected and re-sequenced using the Illumina MiSeq benchtop NGS platform. Sequencing data were analyzed using two different bioinformatic pipelines. Annotated variants were then compared to the previously obtained reference data. Results and conclusions: The analytical sensitivity, specificity and accuracy rates of the Multiplicom CFTR MASTR assay exceeded 99%. Because different types of CFTR mutations can be detected in a single workflow, the CFTR MASTR assay simplifies the overall process and is consequently well suited for routine diagnostics.

scholarly journals Lacer: accurate base quality score recalibration for improving variant calling from next-generation sequencing data in any organism

2017 ◽  
Author(s):  
Jade C.S. Chung ◽  
Swaine L. Chen
Keyword(s):  
Next Generation Sequencing ◽  
Variant Calling ◽  
Quality Score ◽  
Identification Accuracy ◽  
Next Generation Sequencing Data ◽  
Sequencing Error ◽  
Next Generation ◽  
Sequencing Data ◽  
Base Quality Score ◽  
Generation Sequencing

AbstractNext-generation sequencing data is accompanied by quality scores that quantify sequencing error. Inaccuracies in these quality scores propagate through all subsequent analyses; thus base quality score recalibration is a standard step in many next-generation sequencing workflows, resulting in improved variant calls. Current base quality score recalibration algorithms rely on the assumption that sequencing errors are already known; for human resequencing data, relatively complete variant databases facilitate this. However, because existing databases are still incomplete, recalibration is still inaccurate; and most organisms do not have variant databases, exacerbating inaccuracy for non-human data. To overcome these logical and practical problems, we introduce Lacer, which recalibrates base quality scores without assuming knowledge of correct and incorrect bases and without requiring knowledge of common variants. Lacer is the first logically sound, fully general, and truly accurate base recalibrator. Lacer enhances variant identification accuracy for resequencing data of human as well as other organisms (which are not accessible to current recalibrators), simultaneously improving and extending the benefits of base quality score recalibration to nearly all ongoing sequencing projects. Lacer is available at: https://github.com/swainechen/lacer.

scholarly journals cDNA display coupled with next-generation sequencing for rapid activity-based screening: Comprehensive analysis of transglutaminase substrate preference

2021 ◽  
Author(s):  
Jasmina Damnjanović ◽  
Nana Odake ◽  
Jicheng Fan ◽  
Beixi Jia ◽  
Takaaki Kojima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Substrate Preference ◽  
Stable Complex ◽  
Next Generation ◽  
Library Size ◽  
Binary Model ◽  
Wide Range ◽  
Generation Sequencing ◽  
Selection Of

AbstractcDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, where a stable complex is formed suitable for a wide range of selection conditions. A great advantage of cDNA display is the ability to handle enormous library size (1012) in a microtube scale, in a matter of days. To harness its benefits, we aimed at developing a platform which combines the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing (NGS) for the selection of preferred substrate peptides of transglutaminase 2 (TG2), a protein cross-linking enzyme. After the optimization of the platform by the repeated screening of binary model libraries consisting of the substrate and non-substrate peptides at different ratios, screening and selection of combinatorial peptide library randomized at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by NGS and bioinformatics, revealing the comprehensive amino acid preference of the TG2 at targeted positions of the peptide backbone. This is the first report on the cDNA display/NGS screening system to yield comprehensive data on TG substrate preference. Although some issues remain to be solved, this platform can be applied to the selection of other TGs and easily adjusted for the selection of other peptide substrates and even larger biomolecules.

scholarly journals Next-generation sequencing in neuropathological diagnosis of infections of the nervous system

2016 ◽  
Author(s):  
Steven L. Salzberg ◽  
Florian Breitwieser ◽  
Anupama Kumar ◽  
Haiping Hao ◽  
Peter Burger ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Nervous System ◽  
Next Generation Sequencing ◽  
Large Scale ◽  
Infectious Agent ◽  
Next Generation ◽  
Dna And Rna ◽  
Wide Range ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Objective: To determine the feasibility of next-generation sequencing (NGS) microbiome approaches in the diagnosis of infectious disorders in brain or spinal cord biopsies in patients with suspected central nervous system (CNS) infections. Methods: In a prospective-pilot study, we applied NGS in combination with a new computational analysis pipeline to detect the presence of pathogenic microbes in brain or spinal cord biopsies from ten patients with neurological problems indicating possible infection but for whom conventional clinical and microbiology studies yielded negative or inconclusive results. Results: Direct DNA and RNA sequencing of brain tissue biopsies generated 8.3 million to 29.1 million sequence reads per sample, which successfully identified with high confidence the infectious agent in three patients, identified possible pathogens in two more, and helped to understand neuropathological processes in three others, demonstrating the power of large-scale unbiased sequencing as a novel diagnostic tool. Validation techniques confirmed the pathogens identified by NGS in each of the three positive cases. Clinical outcomes were consistent with the findings yielded by NGS on the presence or absence of an infectious pathogenic process in eight of ten cases, and were non-contributory in the remaining two. Conclusions: NGS-guided metagenomic studies of brain, spinal cord or meningeal biopsies offer the possibility for dramatic improvements in our ability to detect (or rule out) a wide range of CNS pathogens, with potential benefits in speed, sensitivity, and cost. NGS-based microbiome approaches present a major new opportunity to investigate the potential role of infectious pathogens in the pathogenesis of neuroinflammatory disorders.

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