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 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 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 reveals two populations of damage-induced small RNAs at endogenous DNA double-strand breaks

2018 ◽  
Vol 46 (22) ◽  
pp. 11869-11882 ◽  
Author(s):  
Franziska Bonath ◽  
Judit Domingo-Prim ◽  
Marcel Tarbier ◽  
Marc R Friedländer ◽  
Neus Visa
Keyword(s):  
Next Generation Sequencing ◽  
Small Rnas ◽  
Double Strand Breaks ◽  
Next Generation ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Two Populations ◽  
Generation Sequencing

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.

scholarly journals Next-generation sequencing of small RNAs from inner ear sensory epithelium identifies microRNAs and defines regulatory pathways

BMC Genomics ◽  
2014 ◽  
Vol 15 (1) ◽  
pp. 484 ◽  
Author(s):  
Anya Rudnicki ◽  
Ofer Isakov ◽  
Kathy Ushakov ◽  
Shaked Shivatzki ◽  
Inbal Weiss ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Inner Ear ◽  
Small Rnas ◽  
Sensory Epithelium ◽  
Next Generation ◽  
Regulatory Pathways ◽  
Generation Sequencing

scholarly journals Next Generation Sequencing Based In-house HIV Genotyping Method: Validation Report

2021 ◽  
Author(s):  
Alisen Ayitewala ◽  
Isaac Ssewanyana ◽  
Charles Kiyaga
Keyword(s):  
Drug Resistance ◽  
Quality Control ◽  
Next Generation Sequencing ◽  
Low Income ◽  
Rna Extraction ◽  
Clinical Samples ◽  
Small Range ◽  
Next Generation ◽  
Genotyping Method ◽  
Generation Sequencing

Abstract BackgroundHIV genotyping has had a significant impact on care and treatment of HIV/AIDS. At clinical level, the test guides physicians on the choice of treatment regimens. At surveillance level, it informs policy on consolidated treatment guidelines and microbial resistance control strategies. Until recently, the conventional test has utilized Sanger sequencing (SS) method. Unlike Next Generation Sequencing (NGS), SS is limited by low data throughput and the inability of detecting low abundant drug resistant variants. NGS has the capacity to improve sensitivity and quantitatively identify low-abundance variants; in addition, it has the potential to improve efficiency as well as lowering costs when samples are batched. Despite the NGS benefits, its utilization in clinical drug resistance profiling is faced with mixed reactions. These are largely based on lack of a consensus regarding the quality control strategy. Nonetheless, transitional views suggest validating the method against the gold-standard SS. Therefore, we present a validation report of an NGS-based in-house HIV genotyping method against SS method in Uganda. ResultsSince there were no established proficiency test panels for NGS-based HIV genotyping, fifteen (15) clinical plasma samples for routine care were utilized. The use of clinical samples allowed for accuracy and precision studies. The workflow involved four (4) main steps; viral RNA extraction, targeted amplicon generation, amplicon sequencing and data analysis. Accuracy of 98% with an average percentage error of 3% was reported for the NGS based assay against the SS platform demonstrating similar performance. The coefficient of variation (CV) findings for both the inter-run and inter-personnel precision showed no variability (CV ≤0%) at the relative abundance of ≥20%. For both inter-run and inter-personnel, variation that affected the precision was observed at 1% frequency. Overall, for all the frequencies, CV registered a small range of (0-2%).Conclusion The NGS-based in-house HIV genotyping method fulfilled the minimum requirements that support its utilization for drug resistance profiling in a clinical setting of a low-income country. For more inclusive quality control studies, well characterized wet panels need to be established.

scholarly journals Factors Impacting Clinically Relevant RNA Fusion Assays Using Next-Generation Sequencing

2021 ◽  
Author(s):  
Nisha S. Ramani ◽  
Keyur P. Patel ◽  
Mark J. Routbort ◽  
Hector Alvarez ◽  
Russell Broaddus ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Core Needle Biopsy ◽  
Needle Biopsy ◽  
Rna Extraction ◽  
Next Generation ◽  
Success Rates ◽  
Detection Rates ◽  
Core Needle ◽  
Rna Quality ◽  
Generation Sequencing

Context.— RNA-based next-generation sequencing (NGS) assays are being used with increasing frequency for comprehensive molecular profiling of solid tumors. Objective.— To evaluate factors that might impact clinical assay performance. Design.— A 4-month retrospective review of cases analyzed by a targeted RNA-based NGS assay to detect fusions was performed. RNA extraction was performed from formalin-fixed, paraffin-embedded tissue sections and/or cytology smears of 767 cases, including 493 in-house and 274 outside referral cases. The types of samples included 422 core needle biopsy specimens (55%), 268 resection specimens (35%), and 77 cytology samples (10%). Results.— Successful NGS fusion testing was achieved in 697 specimens (90.9%) and correlated positively with RNA yield (P < .001) and negatively with specimen necrosis (P = .002), decalcification (P < .001), and paraffin block age of more than 2 years (P = .001). Of the 697 cases that were successfully sequenced, 50 (7.2%) had clinically relevant fusions. The testing success rates and fusion detection rates were similar between core needle biopsy and cytology samples. In contrast, RNA fusion testing was often less successful using resection specimens (P = .007). Testing success was independent of the tumor percentage in the specimen, given that at least 20% tumor cellularity was present. Conclusions.— The success of RNA-based NGS testing is multifactorial and is influenced by RNA quality and quantity. Identification of preanalytical factors affecting RNA quality and yield can improve NGS testing success rates.

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