Search Results (66)

Massively parallel or next-generation sequencing (NGS) has enabled the genetic characterization of cancer patients, allowing the identification of somatic and germline variants associated with their diagnosis, tumor classification, and therapy response. Despite its benefits, NGS testing is not yet available in the Chilean public health system, rendering it both costly and time-consuming for patients and clinicians. Using a retrospective cohort of 67 formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) samples, we aimed to implement the identification, annotation, and prioritization of relevant actionable tumor somatic variants in our laboratory, as part of the public health system. We compared two different library preparation methodologies (amplicon-based and capture-based) and different bioinformatics pipelines for sequencing analysis to assess advantages and disadvantages of each one. We obtained 80.5% concordance between actionable variants detected in our analysis and those obtained in the Cancer Genomics Laboratory from the Universidad de Chile (62 out of 77 variants), a validated laboratory for this methodology. Notably, 98.4% (61 out of 62) of variants detected previously by the validated laboratory were also identified in our analysis. Then, comparing the hybridization capture-based library preparation methodology with the amplicon-based strategy, we found ~94% concordance between identified actionable variants across the 15 shared genes, analyzed by the TumorSec^TM bioinformatics pipeline, developed by the Cancer Genomics Laboratory. Our results demonstrate that it is entirely viable to implement an NGS-based analysis of actionable variant identification and prioritization in cancer samples in our laboratory, being part of the Chilean public health system and paving the way to improve the access to such analyses. Considering the economic realities of most Latin American countries, using a small NGS panel, such as TumorSec^TM, focused on relevant variants of the Chilean and Latin American population is a cost-effective approach to extensive global NGS panels. Furthermore, the incorporation of automated bioinformatics analysis in this streamlined assay holds the potential of facilitating the implementation of precision medicine in this geographic region, which aims to greatly support personalized treatment of cancer patients in Chile. Full article

Background/Objectives: Ancient DNA (aDNA) research workflows heavily depend on efficient aDNA extraction and NGS library preparation. In this study, we compared some of the commonly used laboratory protocols and compared the source of the bone material for sufficient and reliable results. Methods: We executed a three-phase study. First, we analyzed about 2000 previously processed archaic bone samples and conducted a comparative analysis. The second phase involved a controlled experiment of five ancient individuals, with internal control, to further investigate the efficiency of some of the methods. In the third phase, we made a comparison between the efficiency of two enzymes used for library preparation. Results: Samples made from Pars petrosa resulted in the highest yield of endogenous DNA and longer fragment sizes compared to tooth or skeletal samples. DNA extraction made by MinElute columns preserved slightly longer fragments than the handmade silica suspension. NGS libraries indexed using AccuPrime Pfx produced slightly more consistent insert sizes compared to GoTaq G2. Samples prepared with GoTaq G2 contained slightly more unique molecules. The duplication rates showed no significant impact from enzyme choice. Conclusions: Pars petrosa remains the most reliable source of aDNA, with the extraction method using MinElute columns. While AccuPrime Pfx ensures precise NGS library preparation, a more economical choice of the GoTaq G2 enzyme is a viable alternative for degraded archaic samples. Full article

SARS-CoV-2 high transmission and genomic mutations result in the emergence of new variants that impact COVID-19 vaccine efficacy and virus transmission by evading the host immune system. Wastewater-based epidemiology is an effective approach to monitor SARS-CoV-2 variants circulation in the population but is a challenge due to the presence of reaction inhibitors and the low concentrations of SARS-CoV-2 in this environment. Here, we aim to improve SARS-CoV-2 variant detection in wastewater by employing nested PCR followed by next-generation sequencing (NGS) of small amplicons of the S gene. Eight SARS-CoV-2 wastewater samples from Alegria Wastewater Treatment Plant, in Rio de Janeiro, Brazil, were collected monthly from February to September 2021. Samples were submitted to virus concentration, RNA extraction and nested PCR followed by NGS. The small amplicons were used to prepare libraries for sequencing without the need to perform any fragmentation step. We identified and calculated the frequencies of 29 mutations matching the Alpha, Beta, Gamma, Delta, Omicron, and P.2 variants. Omicron matching-mutations were detected before the lineage was classified as a variant of concern. SARS-CoV-2 wastewater sequences clustered with SARS-CoV-2 variants detected in clinical samples that circulated in 2021 in Rio de Janeiro. We show that sequencing of selected small amplicons of SARS-CoV-2 S gene allows the identification of SARS-CoV-2 variants matching mutations and their frequencies’ calculation. This approach may be expanded using customizing primers for additional genomic regions, in order to differentiate current variants. Approaches that allow us to learn how variants emerge and how they relate to clinical outcomes are crucial for our understanding of the dynamics of virus variants circulation, providing valuable data for public health management. Full article

Zearalenone (ZEN), a toxic estrogenic mycotoxin in cereals, threatens human and animal health through reproductive, immune, and cytotoxic effects, necessitating sensitive detection methods. While nanobodies offer advantages over conventional antibodies for on-site ZEN detection, their application remains unexplored. This study aimed to develop an anti-ZEN nanobody derived from an anti-ZEN phage display nanobody library. An alpaca was immunized with a ZEN-bovine serum albumin (ZEN-BSA) antigen, achieving peak serum antibody titers (1:25,600) following four immunizations. A high-capacity phage display nanobody library (1.0 × 10¹¹ plaque-forming units/mL) was constructed. Following four rounds of biopanning, an enrichment factor of 479 was achieved. Phage ELISA screening identified six phage display nanobodies with specific ZEN-binding activity, and multiple sequence alignment revealed four unique nanobody sequences. The selected phage display nanobody, designated phage-V44, was expressed and purified, and its presence was validated by SDS-PAGE and western blotting, which detected a single approximately 17 kDa band consistent with the expected nanobody size. We established a working curve for an indirect competitive enzyme-linked immunoassay (ELISA) for ZEN, which showed an IC50 value of 7.55 ng/mL. The specificity and affinity of the V44 were also verified. Collectively, the study successfully constructed an anti-ZEN phage display nanobody library, screened four specific ZEN-binding phage display nanobodies, and prepared the anti-ZEN nanobody V44. Thereby establishing a foundation for the nanobody’s future integration into rapid on-site detection methods for ZEN in both animal feed and human food products. Full article

Background: Analysing circulating miRNAs in paediatric plasma is challenging due to typically low sample volumes. The QIAseq miRNA UDI Library Kit (Qiagen, Hilden, Germany) was selected as it has a proven track record with a specific protocol for plasma and serum. The protocol, however, required optimisation for use with low-volume paediatric plasma samples before generating acceptable yields in our cohort. Methods: The miRNeasy Serum/Plasma kit (Qiagen) and the MagMAX miRVana Total Isolation kit (ThermoFisher Scientific, Waltham, MA, USA) were assessed following the manufacturer’s instructions with 100 µL and 200 µL of paediatric plasma. Libraries were prepared using the QIAseq miRNA UDI Library Kit (Qiagen). Optimisations were made for the QIAseq miRNA UDI Library Kit (Qiagen) using total RNA extracted with the miRNeasy Serum/Plasma kit (Qiagen) from 100 µL of plasma. Results: Prior to optimisation, both RNA extraction kits underperformed with the QIAseq miRNA UDI Library kit, producing low miRNA library yields ranging between 0 and 1.42 ng/µL. Plasma input volumes of 100 µL and 200 µL demonstrated no significant differences. Adjusting the QIAseq protocol for low RNA concentrations improved miRNA library yields, an average of 5.6 ng/µL and a maximum of 24.3 ng/µL across 92 samples. The optimised protocol showed no age or gender biases with the QIAseq kit. Conclusions: Failure rates in miRNA library preparations are rarely reported, making it hard to gauge whether the 8.7% failure rate observed here is typical. However, given the challenges of using low-concentration, low-volume paediatric plasma, this represents a significant improvement over previous attempts, supporting further research in the field. Full article

It has been shown that the pathogenic variants (PVs) of the DNA Damage Response (DDR) genes, whether of a germinal or somatic nature, represent a predictive biomarker of high sensitivity to treatment with inhibitors of the enzyme poly-ADP-ribose polymerase (PARP) in patients with hormone-resistant metastatic prostate cancer (HRPCa). Moreover, the detection of PVs of the Homologous Recombination Repair (HRR) genes in PCa patients can help to define the patient’s prognosis and the choice of the therapeutic procedure. Among men with metastatic PCa, the frequency of PVs in HRR genes ranges from 11% to 33%, which is a significantly higher rate compared to non-metastatic PCa, where the incidence is between 5% and 10%. Next-Generation Sequencing (NGS) results were more commonly obtained from newly acquired somatic samples compared to archived samples (prostate biopsy or prostatectomy). We developed an experimental multidisciplinary prospective study in patients with a new diagnosis of high-risk PCa at biopsy. The aim was to evaluate the presence of PVs of different HRR genes in patients with the first diagnosis of PCa in relation to a metastatic or non-metastatic stage, tumor aggressiveness, and early risk of progression. Among 43 initial tumor samples from 22 patients, 25 samples from 12 patients were selected for library preparation based on their DNA concentration and quality. After the NGS, 14 different DNA variants were prioritized. Oncogenetic and likely oncogenetic variants were found in the ATM, BRCA1, PTEN, KMT2D, and CDH1 genes. Moreover, variants of uncertain significance were found in ATM, DDR2, FANCA, FOXA1, PLCB4, PTCH1, and RB1. Full article

Background/Objectives: The nucleic acid-based product (NAP) portfolio is expanding continuously and provides safer curative options for many disease indications. Nucleic acid-based products offer several advantages compared to proteins and virus-based products. They represent an emerging field; thus, their quality control and regulatory landscape is evolving to ensure adequate quality and safety. Next-Generation Sequencing (NGS) is mostly recommended for NAP identity testing, and we are leveraging its application for impurity profiling. Methods: We proposed a workflow for the purity assessment of NAPs through short-read Illumina NGS followed by data analysis of mRNA vaccine and pDNA samples. We determined the sequence identity, DNA and RNA contamination, off-target RNA contamination, and poly-A count with the proposed workflow. Results: Our workflow predicted most of the critical quality controls of mRNA vaccine and plasmid DNA samples, especially focusing on the identity and the nucleotide-based impurities. Additionally, NGS data interpretation also assisted in strategic decisions for NAP manufacturing process optimizations. Conclusions: We recommend the adaptation of incremental NGS data by regulatory agencies to identify nucleotide-based impurities in NAPs. Perhaps NGS adaptation under cGMP compliance needs to be deliberated with the regulatory bodies, especially focusing on the methods qualification and validation part, starting from the sample collection, NGS library preparation, NGS run, and its data analysis pipeline. Full article

Background: Comprehensive genomic profiling (CGP) has gained an important role in patients with advanced prostate cancer following the introduction of PARP inhibitors in daily clinical practice. Here, we report an overview of CGP results, specifically of BRCA1 and BRCA2 HRD-repair system genes, from patients with prostate cancer analyzed in our institution, and we compare our results with those available from more recent scientific literature. Methods: The study cohort consisted of 70 patients. Somatic DNA was extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue using a MagCore Genomic DNA FFPE One-Step Kit for MagCore System. The DNA was quantified by EasyPGX^® Real-Time qPCR and EasyPGX^® Analysis Software (version 4.0.13). Tissue somatic DNA libraries were prepared with Myriapod^® NGS BRCA1-2 panel-NG035 and sequenced in a Mi-Seq^® System. The sequence alignment in hg19 and the variant calling were performed using Myriapod^® NGS Data Analysis Software version 5.0.8 NG900-SW 5.0.8 with a software detection limit (LoD) of 95%. Variants with a coverage of 500 and VAF% ≥ 5 were evaluated. Results: Tumor tissue NGS was unsuccessful in 46/70 patients (66%). Mutations of the BRCA2 gene were detected in 4 of the samples: (1) BRCA2 ex10 c.1244A>G p.His415Arg VAF = 51.03%; (2) BRCA2 ex11 c.5946delT p.Ser1982fs VAF = 72.1%; (3) BRCA2 ex11 c.3302A>G p.His1101Arg VAF = 52.9%; and (4) BRCA2 ex11 c.3195_3198delTAAT p.Asn1066fs VAF = 51.1%. Conclusions: The results from our internal overview seem to support the data and to confirm the performance of the technical issues reported in the literature. Considering the advanced age of our patients, with 84% of men over the age of 65, the application of alternative and less invasive procedures such as liquid biopsy, could be a more suitable solution for some cases. Full article

All subtypes of Clostridium perfringens (C. perfringens) produce the alpha toxin (CPA), which can cause enteritis or enterotoxemia in lambs, cattle, pigs, and horses, as well as traumatic clostridial myonecrosis in humans and animals. CPA acts on cell membranes, ultimately leading to endocytosis and cell death. Therefore, the neutralization of CPA is crucial for the prevention and treatment of diseases caused by C. perfringens. In this study, utilizing CPA as an antigen, a nanobody (CPA-VHH) with a half-life of 2.9 h, an affinity constant (KD) of 0.9 nmol/L, and good stability below 60 °C was prepared from a natural nanobody library from alpacas. The biological activity analysis of CPA-VHH revealed its ability to effectively neutralize the phospholipase and hemolytic activity of CPA at a 15-fold ratio. In Vero cells, 9.8 μg/mL CPA-VHH neutralized the cytotoxicity of CPA at two times the half-maximal inhibitory concentration (IC₅₀). In a mouse model, 35.7 ng/g body weight (BW) of CPA-VHH neutralized 90% of the lethality caused by a 2× median lethal dose (LD₅₀) of CPA. It was found that CPA-VHH protected 80% of mice within 30 min at 2 × LD₅₀ CPA, but this dropped below 50% after 2 h and to 0% after 4 h. Rescue trials indicated that using CPA-VHH within 30 min post-infection with 2 × LD₅₀ CPA achieved an 80% rescue rate, which decreased to 10% after 2 h. Furthermore, CPA-VHH effectively mitigated the reduction in the expression levels of zonula occludens-1 (ZO-1), Occludin, and Claudin-1, while also attenuating the upregulation of the pro-inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), tumor necrosis factor α (TNF-α), and interferon-γ (IFN-γ) induced by CPA infection. Overall, this study has identified a specific nanobody, CPA-VHH, that effectively neutralizes CPA toxins in vitro and in animal models, providing a new tool for inhibiting the pathogenicity resulting from these toxins and laying an important foundation for the development of new anti-C. perfringens toxin-related therapeutic products. Full article

Background: Multidrug-resistant HIV strains challenge treatment efficacy and increase mortality rates. Next-generation sequencing (NGS) technology swiftly detects variants, facilitating personalized antiretroviral therapy. Aim: This study aimed to validate the Vela Diagnostics NGS platform for HIV drug resistance mutation analysis, rigorously assessed with clinical samples and CAP proficiency testing controls previously analyzed by Sanger sequencing. Method: The experimental approach involved the following: RNA extraction from clinical specimens, reverse transcription polymerase chain reaction (RT-PCR) utilizing the Sentosa SX 101 platform, library preparation with the Sentosa SQ HIV Genotyping Assay, template preparation, sequencing using the Sentosa SQ301 instrument, and subsequent data analysis employing the Sentosa SQ Suite and SQ Reporter software. Drug resistance profiles were interpreted using the Stanford HIV Drug Resistance Database (HIVdb) with the HXB2 reference sequence. Results: The Vela NGS system successfully identified a comprehensive array of drug resistance mutations across the tested samples: 28 nucleoside reverse transcriptase inhibitors (NRTI), 25 non-nucleoside reverse transcriptase inhibitors (NNRTI), 25 protease inhibitors (PI), and 10 integrase gene-specific variants. Dilution experiments further validated the system’s sensitivity, detecting drug resistance mutations even at viral loads lower than the recommended threshold (1000 copies/mL) set by Vela Diagnostics. Scope: This study underscores the validation and clinical applicability of the Vela NGS system, and its implementation may offer clinicians enhanced precision in therapeutic decision-making for individuals living with HIV. Full article

Acute respiratory diseases in felines can be attributed to a diverse range of pathogens. The recent emergence of novel viruses, particularly SARS-CoV-2 and its variants, has also been associated with respiratory ailments in cats and other pets, underscoring the need for a highly sensitive diagnostic assay capable of concurrently detecting multiple respiratory pathogens. In this study, we developed a targeted next generation sequencing panel using Ion Torrent Ampliseq technology to detect multiple respiratory pathogens, including recent SARS-CoV-2 variants and Feline herpesvirus-1, Feline calicivirus, Bordetella bronchiseptica, Mycoplasmopsis (previously Mycoplasma) felis, and Chlamydia felis. A PCR amplification-based library preparation, employing primers designed for pathogen target regions, was synthesized and divided into two pools, followed by sequencing and assembly to a repertoire of target pathogen genomes. Analytical sensitivity was assessed based on Ct values from real-time PCR for the corresponding pathogens, indicating an equivalent detection limit. Most of the pathogens under study were positively identified to a limit of approximately Ct 36, whereas for Feline herpesvirus-1 and SARS-CoV-2, positive reads were observed in samples with a Ct of 37. Based on a limited number of samples, the diagnostic sensitivity values for the SARS-CoV-2, Feline herpesvirus-1, and M. felis samples were 100% with no false negative results. The diagnostic specificity of SARS-CoV-2, Feline herpesvirus-1, Feline calicivirus, and C. felis were 100%. Importantly, none of the target primers exhibited non-specific amplification, ensuring the absence of false positive results for other pathogens within the study. Additionally, the assay’s specificity was validated by cross-referencing the raw sequencing data with established databases like BLAST, affirming the high specificity of the targeted Next-Generation Sequencing (tNGS) assay. Variations in the sequencing reads of different pathogens were observed when subjected to diverse extraction methods. Rigorous assessment of the assay’s reliability involved reproducibility across testing personnel and repeated runs. The developed assay’s clinical applicability was tested using samples submitted to the diagnostic laboratory from cat shelters and suspected cases. The developed targeted next-generation sequencing methodology empowers the detection of multiple respiratory pathogens manifesting similar clinical symptoms while offering confirmation of results through genome sequencing. Full article

Background: MicroRNA (miRNA) have the potential to be non-invasive and attractive biomarkers for a vast number of diseases and clinical conditions; however, a reliable analysis of miRNA expression in blood samples meets a number of methodological challenges. In this report, we presented and discussed, specifically, the principles and limitations of miRNA purification and analysis in blood plasma samples collected from the left atrium during an ablation procedure on patients with atrial fibrillation (AF). Materials and Methods: Consecutive patients hospitalized in the First Department of Cardiology for pulmonary vein ablation were included in this study (11 with diagnosed paroxysmal AF, 14 with persistent AF, and 5 without AF hospitalized for left-sided WPW ablation—control group). Whole blood samples were collected from the left atrium after transseptal puncture during the ablation procedure of AF patients. Analysis of the set of miRNA molecules was performed in blood plasma samples using the MIHS-113ZF-12 kit and miScript microRNA PCR Array Human Cardiovascular Disease. Results: The miRNS concentrations were in the following ranges: paroxysmal AF: 7–23.1 ng/µL; persistent AF: 4.9–66.8 ng/µL; controls: 6.3–10.6 ng/µL. The low A260/280 ratio indicated the protein contamination and the low A260/A230 absorbance ratio suggested the contamination by hydrocarbons. Spectrophotometric measurements also indicated low concentration of nucleic acids (<10 ng/µL). Further steps of analysis revealed that the concentration of cDNA after the Real-Time PCR (using the PAXgene RNA Blood kit) reaction was higher (148.8 ng/µL vs. 68.4 ng/µL) and the obtained absorbance ratios (A260/A280 = 2.24 and A260/A230 = 2.23) indicated adequate RNA purity. Conclusions: Although developments in miRNA sequencing and isolation technology have improved, detection of plasma-based miRNA, low RNA content, and sequencing bias introduced during library preparation remain challenging in patients with AF. The measurement of the quantity and quality of the RNA obtained is crucial for the interpretation of an efficient RNA isolation. Full article

Next-generation sequencing (NGS) tools have importantly helped the classification of myelodysplastic syndromes (MDS), guiding the management of patients. However, new concerns are under debate regarding their implementation in routine clinical practice for the identification of germline predisposition. Cost-effective targeted NGS tools would improve the current standardized studies and genetic counseling. Here, we present our experience in a preliminary study detecting variants using a two-time multiplexed library strategy. Samples from different MDS patients were first mixed before library preparation and later multiplexed for a sequencing run. Two different mixes including a pool of three (3×) and four (4×) samples were evaluated. The filtered variants found in the individually sequenced samples were compared with the variants found in the two-time multiplexed studies to determine the detection efficiency scores. The same candidate variants were found in the two-time multiplexed studies in comparison with the individual tNGS. The variant allele frequency (VAF) values of the candidate variants were also compared. No significant differences were found between the expected and observed VAF percentages in both the 3× (p-value 0.74) and 4× (p-value 0.34) multiplexed studies. Our preliminary results suggest that the two-time multiplexing strategy might have the potential to help reduce the cost of evaluating germline predisposition. Full article

In recent years, high-throughput sequencing methods have become increasingly popular in molecular biology laboratories, mainly due to the relatively low cost of small, benchtop platforms, the simplicity of library preparation, and the low price per unit of information. Sequencing huge and complex genomes, such as cereal genomes, remains challenging and may not always be necessary. Therefore, several techniques have been developed to sequence a reduced representation of the genome. The most flexible and widely used of these is ddRAD-Seq, which uses a pair of restriction enzymes to generate a pool of DNA fragments. The aim of this study was to validate in vitro the efficacy of different combinations of restriction enzymes for ddRAD-Seq library construction in barley and maize. Eleven pairs of restriction enzymes were selected and tested to determine the concentrations of fragments with the expected length range and to select suitable pairs for sampling the genomes of these two cereals using ddRAD-Seq. For the selected pairs, i.e., PstI—MspI and HindIII—FspBI for barley and maize, respectively, libraries were prepared for NGS sequencing on Illumina MiSeq. Sequencing confirmed the suitability of the selected enzymes to perform ddRAD-Seq in different genotypes. The results presented can be used for extensive research on these important cereal species. Full article

Detecting copy number variations (CNVs) and alterations (CNAs) in the BRCA1 and BRCA2 genes is essential for testing patients for targeted therapy applicability. However, the available bioinformatics tools were initially designed for identifying CNVs/CNAs in whole-genome or -exome (WES) NGS data or targeted NGS data without adaptation to the BRCA1/2 genes. Most of these tools were tested on sample cohorts of limited size, with their use restricted to specific library preparation kits or sequencing platforms. We developed BRACNAC, a new tool for detecting CNVs and CNAs in the BRCA1 and BRCA2 genes in NGS data of different origin. The underlying mechanism of this tool involves various coverage normalization steps complemented by CNV probability evaluation. We estimated the sensitivity and specificity of our tool to be 100% and 94%, respectively, with an area under the curve (AUC) of 94%. The estimation was performed using the NGS data obtained from 213 ovarian and prostate cancer samples tested with in-house and commercially available library preparation kits and additionally using multiplex ligation-dependent probe amplification (MLPA) (12 CNV-positive samples). Using freely available WES and targeted NGS data from other research groups, we demonstrated that BRACNAC could also be used for these two types of data, with an AUC of up to 99.9%. In addition, we determined the limitations of the tool in terms of the minimum number of samples per NGS run (≥20 samples) and the minimum expected percentage of CNV-negative samples (≥80%). We expect that our findings will improve the efficacy of BRCA1/2 diagnostics. BRACNAC is freely available at the GitHub server. Full article