Application Of Next-generation Sequencing Research Articles

Application of metagenomic next-generation sequencing in optimizing the diagnosis of ascitic infection in patients with liver cirrhosis

BackgroundMetagenomic next-generation sequencing (mNGS) is an emerging technique for the clinical diagnosis of infectious disease that has rarely been used for the diagnosis of ascites infection in patients with cirrhosis. This study compared mNGS detection with conventional culture methods for the on etiological diagnosis of cirrhotic ascites and evaluated the clinical effect of mNGS.MethodsA total of 109 patients with ascites due to cirrhosis were included in the study. We compared mNGS with conventional culture detection by analyzing the diagnostic results, pathogen species and clinical effects. The influence of mNGS on the diagnosis and management of ascites infection in patients with cirrhosis was also evaluated.ResultsAscites cases were classified into three types: spontaneous bacterial peritonitis (SBP) (16/109, 14.7%), bacterascites (21/109, 19.3%) and sterile ascites (72/109, 66.1%). In addition, 109 patients were assigned to the ascites mNGS-positive group (80/109, 73.4%) or ascites mNGS-negative group (29/109, 26.6%). The percentage of positive mNGS results was significantly greater than that of traditional methods (73.4% vs. 28.4%, P < 0.001). mNGS detected 43 strains of bacteria, 9 strains of fungi and 8 strains of viruses. Fourteen bacterial strains and 3 fungal strains were detected via culture methods. Mycobacteria, viruses, and pneumocystis were detected only by the mNGS method. The mNGS assay produced a greater polymicrobial infection rate than the culture method (55% vs. 16%). Considering the polymorphonuclear neutrophil (PMN) counts, the overall percentage of pathogens detected by the two methods was comparable, with 87.5% (14/16) in the PMN ≥ 250/mm3 group and 72.0% (67/93) in the PMN < 250/mm3 group (P > 0.05). Based on the ascites PMN counts combined with the mNGS assay, 72 patients (66.1%) were diagnosed with ascitic fluid infection (AFI) (including SBP and bacterascites), whereas based on the ascites PMN counts combined with the culture assay, 37 patients (33.9%) were diagnosed with AFI (P < 0.05). In 60 (55.0%) patients, the mNGS assay produced positive clinical effects; 40 (85.7%) patients had their treatment regimen adjusted, and 48 patients were improved. The coincidence rate of the mNGS results and clinical findings was 75.0% (60/80).ConclusionsCompared with conventional culture methods, mNGS can improve the detection rate of ascites pathogens, including bacteria, viruses, and fungi, and has significant advantages in the diagnosis of rare pathogens and pathogens that are difficult to culture; moreover, mNGS may be an effective method for improving the diagnosis of ascites infection in patients with cirrhosis, guiding early antibiotic therapy, and for reducing complications related to abdominal infection. In addition, explaining mNGS results will be challenging, especially for guiding the treatment of infectious diseases.

Clinical applications of metagenomic next-generation sequencing in the identification of pathogens in periprosthetic joint infections: a retrospective study

BackgroundThis study aimed to evaluate the application of metagenomic next-generation sequencing (mNGS) technology to identify pathogens in periprosthetic joint infection (PJI).MethodsA retrospective analysis was conducted on 65 patients suspected of having PJI between April 2020 and July 2023. The patients were categorized into PJI (46 patients) and non-PJI (19 patients) groups based on the 2018 International Consensus Meeting criteria. Clinical data were collected, and both conventional bacterial culture and mNGS were performed. The diagnostic performance of the two methods was compared and analyzed.ResultsmNGS exhibited a sensitivity of 89.13%, a specificity of 94.74%, a positive predictive value of 97.62%, a negative predictive value of 78.26%, and an overall diagnostic accuracy of 90.77%. Compared to microbial culture, mNGS demonstrated superior diagnostic sensitivity while maintaining similar specificity. A total of 48 pathogens were successfully identified using mNGS, with Coagulase-negative staphylococci, Streptococci, Staphylococcus aureus, and Cutibacterium acnes being the most common infectious agents. Notably, mNGS was used to identify 17 potential pathogens in 14 culture-negative PJI samples, highlighting its ability to detect rare infectious agents, including Cutibacterium acnes (n = 5), Granulicatella adiacens (n = 1), Mycobacterium tuberculosis complex (n = 1), and Coxiella burnetii (n = 1), among others, which are not detectable by routine culture methods. However, mNGS failed to detect the pathogen in 4 culture-positive PJI patients, indicating its limitations. Among the 46 PJI patients, 27 had positive culture and mNGS results. The results of mNGS were concordant with those of culture at the genus level in 6 patients with PJI and at the species level in 18 patients. Furthermore, the present study revealed a significantly greater proportion of Staphylococcus aureus in the sinus tract group (45.45%) than in the non-sinus tract group (14.29%), indicating the association of this pathogen with sinus formation in PJI (P = 0.03). Additionally, there was no significant difference in the occurrence of polymicrobial infections between the sinus tract group (27.27%) and the non-sinus tract group (33.33%) (P = 0.37).ConclusionsMetagenomic next-generation sequencing can serve as a valuable screening tool in addition to traditional culture methods to improve diagnostic accuracy through optimized culture strategies.

Application of Metagenomic Next-Generation Sequencing in the Diagnosis of Infectious Keratitis.

To determine the advantages of next-generation metagenomic sequencing (mNGS) technology in the diagnosis and treatment of infectious keratitis (IK). A total of 287 patients with IK admitted to the Department of Ophthalmology of Nanjing First Hospital between August 2018 and December 2022 were analyzed retrospectively, and the pathogenic causes, etiological characteristics, detection, treatment methods, and efficacy were summarized. Trauma and foreign matter were the most common causes of IK (144 patients, 50.2%). Of the 287 patients, 228 (79.4%) were diagnosed with a specific etiology, including 110 (48.2%) fungal infections, 44 (19.3%) viral infections, 42 (18.4%) mixed infections, and 30 (13.2%) bacterial infections. Filamentous fungi represented by Fusarium and Aspergillus were the most common, followed by bacteria such as Pseudomonas aeruginosa, Streptococcus pneumoniae, viruses (Herpes Simplex Virus/Varicella-Zoster Virus), and parasites. The positivity rates of secretion culture, corneal laser confocal microscopy (CM), mNGS, and pathological sections were 47.3% (133/281), 45.3% (111/245), 83.9% (104/124), and 19.3% (40/207), respectively. The positivity rate of mNGS for bacteria and viruses was higher than that of the other methods, and the positivity rate for fungi was the same as that for CM. As a result, 214 cases (74.6%) were cured, 51 cases (17.8%) improved, 8 cases (2.8%) did not heal, ocular content enucleation was performed in 14 cases (4.9%), and the overall efficacy rate was 92.3%. Trauma and foreign matter are the main causes of IK. The mNGS technology is an efficient and comprehensive detection method for viruses and bacteria, especially for mixed infections.

Primary immune regulatory disorders (PIRD): expanding the mutation spectrum in Turkey and identification of sixteen novel variants.

Human Inborn Errors of Immunity (IEIs) encompass a clinically and genetically heterogeneous group of disorders, ranging from mild cases to severe, life-threatening types. Among these, Primary Immune Regulatory Disorders (PIRDs) constitute a subset of IEIs characterized by diverse clinical phenotypes, prominently featuring severe atopy, autoimmunity, lymphoproliferation, hyperinflammation, autoinflammation, and susceptibility to malignancies. According to the latest report from the International Union of Immunological Societies (IUIS), PIRDs arise from mutations in various genes including LYST, RAB27A, AP3B1, AP3D1, PRF1, UNC13D, STX11, STXBP2, FAAP24, SLC7A7, RASGRP1, CD70, CTPS1, RLTPR, ITK, MAGT1, PRKCD, TNFRSF9, SH2DIA, XIAP, CD27 (TNFRSF7), FAS (TNFRSF6), FASLG (TNFSF6), CASP10, CASP8, FADD, LRBA, STAT3, AIRE, ITCH, ZAP70, TPP2, JAK1, PEPD, FOXP3, IL2RA, CTLA4, BACH2, IL2RB, DEF6, FERMT1, IL10, IL10RA, IL10RB, NFAT5, TGFB1, and RIPK1 genes. We designed a targeted next-generation sequencing (TNGS) workflow using the Ion AmpliSeq™ Primary Immune Deficiency Research Panel to sequence 264 genes associated with IEIs on the Ion S5™ Sequencer. In this study, we report the identification of 38 disease-causing variants, including 16 novel ones, detected in 40 patients across 15 distinct PIRD genes. The application of next-generation sequencing enabled rapid and precise diagnosis of patients with PIRDs.

Application of metagenomic next-generation sequencing in the diagnosis of Bartonella neuroretinitis: a case report and literature review.

Cat-scratch disease (CSD) is caused by Bartonella henselae infection. In atypical cases of CSD, pathogen determination is challenging. We report a case of Bartonella neuroretinitis with neither a clear history of scratches nor typical general symptoms. The diagnosis was made using metagenomic next-generation sequencing (mNGS), a high-throughput sequencing technology. A female patient presented to the ophthalmologist with complaint of blurred vision in her right eye. Although with history of raising a cat, she reported no clear history of scratches or typical general symptoms, except a fever of unknown origin which resolved spontaneously. The best corrected visual acuity (BCVA) of the right eye was count fingers. Fundus examination showed optic disc oedema, macular exudates and inferior exudative retinal detachment. Laboratory examination results showed increased value of serum C-reactive protein and erythrocyte sedimentation rate. Ocular involvement of toxoplasmosis, syphilis and tuberculosis were excluded. To identify the possible causative pathogen of the disease, mNGS of aqueous humour sample was performed and 521 reads of B. henselae were identified. Serological test results further showed a positive immunoglobulin G (IgG) titre of 1:64. Taking the contact history, clinical manifestations, mNGS and serological results into consideration, the diagnosis of Bartonella neuroretinitis (ocular CSD) was made. After appropriatetreatment, the BCVA of the right eye improved to 20/25 in the last follow-up. Fundus examination showed a normal optic disc and macula, and the exudates had reduced. mNGS, a fast and unbiased method, can be used to detect B. henselae (if present) in intraocular fluid samples.; however, the results should be interpreted together with the clinical symptoms and other auxiliary test results.

Clinical application of metagenomic next-generation sequencing for the diagnosis of suspected infection in adults: A cross-sectional study.

Metagenomic next-generation sequencing (mNGS) has become an available method for pathogen detection. The clinical application of mNGS requires further evaluation. We conducted a cross-sectional study of 104 patients with suspected infection between May 2019 and May 2021. The risk factors associated with infection were analyzed using univariate logistic analysis. The diagnostic performance of pathogens was compared between mNGS and conventional microbiological tests. About 104 patients were assigned into 3 groups: infected group (n = 69), noninfected group (n = 20), and unknown group (n = 15). With the composite reference standard (combined results of all microbiological tests, radiological testing results, and a summary of the hospital stay of the patient) as the gold standard, the sensitivity, specificity, positive predictive value, negative predictive value of mNGS was 84.9%, 50.0%, 88.6%, and 42.1%, respectively. Compared with conventional microbiological tests, mNGS could detect more pathogens and had obvious advantages in Mycobacterium tuberculosis, Aspergillus, and virus detection. Moreover, mNGS had distinct benefits in detecting mixed infections. Bacteria-fungi-virus mixed infections were the most common in patients with severe pneumonia. mNGS had a higher sensitivity than conventional microbiological tests, especially for M. tuberculosis, Aspergillus, viruses, and mixed infections. We suggest that mNGS should be used more frequently in the early diagnosis of pathogens in critically ill patients in the future.

Standardization of next-generation sequencing for detecting mutations associated with targeted therapy and immunotherapy based on dynamic pattern of expandable detection range

Next-generation sequencing (NGS) has laid the foundation for precision oncology care. NGS technologynot only represents an innovation in the methodology but also brings about a revolution in the concept of detecting gene alterations for targeted therapy and immunotherapy of cancers. As basic biomedical research and drug development progress, the landscape of biomarkers associated with gene alterations continues to evolve. Thus, the standardization of NGS-based gene alterations detection should take into account the characteristics of NGS methods and the gene alteration biomarkers. To be specific, whether employed as in vitro diagnostic products or laboratory-developed tests, the detection range can be expanded in response to changes in the clinical evidence level of biomarkers during the process of assay development and clinical application. Such adjustment needs the analytical validation results for supplemented genes or mutant sites within a predefined detection system, which will maximally fulfill the evolving clinical demands in cancer diagnosis and treatment, simultaneously mitigate potential risks effectively. This article primarily discusses the standardization pathway for NGS testing of gene alterations in cancer by focusing on the characteristics of NGS methods, gene alteration biomarkers, and the current status of the standardization of NGS application.

Screening for thalassemia carriers among the Han population of childbearing age in Southwestern of China.

Thalassemia is a severe hereditary blood disorder that poses a significant threat to human health and leads to mortality and disability. It is one of the most prevalent monogenic diseases worldwide. The aim of this study was to analyze the molecular epidemiological data of individuals of childbearing age from the Han ethnic group with thalassemia in Southwest China and to explore the application of next-generation sequencing (NGS) technology in screening thalassemia carriers. The participants were Han males and females of childbearing age who sought medical advice at the West China Second University Hospital, Sichuan University from June 2022 to June 2023. We detected α- and β-thalassemia mutations using full-length capture of the thalassemia genes and NGS technology. In a cohort of 1,093 participants, 130 thalassemia carriers were identified, with an overall detection rate of 11.89% (130/1,093). Among these, 0.91% (10/1,093) had mutations that could not be detected using traditional PCR techniques. The proportions of carriers with α-, β-, and α-complexed β-thalassemia gene mutations were 7.68% (84/1,093), 3.93% (43/1,093), and 0.27% (3/1,093), respectively. We identified a novel HBA2 c.166del variant that has not been previously reported. Using NGS technology, we found that the mutation-carrying rate of thalassemia genes was 11.89% in the Han population of childbearing age in Southwest China. Compared with the results of traditional PCR techniques, NGS detected an additional 0.91% (10/1,093) rare genetic variants. NGS technology should be utilized as the primary screening method for thalassemia carriers among Han nationality people of childbearing age in Southwest China.

Multinational experience with next-generation sequencing: opportunity to identify transthyretin cardiac amyloidosis and Fabry disease.

Sarcomeric hypertrophic cardiomyopathy (HCM) must be differentiated from phenotypically similar conditions because clinical management and prognosis may greatly differ. Patients with unexplained left ventricular hypertrophy require an early, confirmed genetic diagnosis through diagnostic or predictive genetic testing. We tested the feasibility and practicality of the application of a 17-gene next-generation sequencing (NGS) panel to detect the most common genetic causes of HCM and HCM phenocopies, including treatable phenocopies, and report detection rates. Identification of transthyretin cardiac amyloidosis (ATTR-CA) and Fabry disease (FD) is essential because of the availability of disease-specific therapy. Early initiation of these treatments may lead to better clinical outcomes. In this international, multicenter, cross-sectional pilot study, peripheral dried blood spot samples from patients of cardiology clinics with an unexplained increased left ventricular wall thickness (LVWT) of ≥13 mm in one or more left ventricular myocardial segments (measured by imaging methods) were analyzed at a central laboratory. NGS included the detection of known splice regions and flanking regions of 17 genes using the Illumina NextSeq 500 and NovaSeq 6000 sequencing systems. Samples for NGS screening were collected between May 2019 and October 2020 at cardiology clinics in Colombia, Brazil, Mexico, Turkey, Israel, and Saudi Arabia. Out of 535 samples, 128 (23.9%) samples tested positive for pathogenic/likely pathogenic genetic variants associated with HCM or HCM phenocopies with double pathogenic/likely pathogenic variants detected in four samples. Among the 132 (24.7%) detected variants, 115 (21.5%) variants were associated with HCM and 17 (3.2%) variants with HCM phenocopies. Variants in MYH7 (n=60, 11.2%) and MYBPC3 (n=41, 7.7%) were the most common HCM variants. The HCM phenocopy variants included variants in the TTR (n=7, 1.3%) and GLA (n=2, 0.4%) genes. The mean (standard deviation) ages of patients with HCM or HCM phenocopy variants, including TTR and GLA variants, were 42.8 (17.9), 54.6 (17.0), and 69.0 (1.4) years, respectively. The overall diagnostic yield of 24.7% indicates that the screening strategy effectively identified the most common forms of HCM and HCM phenocopies among geographically dispersed patients. The results underscore the importance of including ATTR-CA (TTR variants) and FD (GLA variants), which are treatable disorders, in the differential diagnosis of patients with increased LVWT of unknown etiology.

Abstract 2298: Optimization of high-volume cell free DNA extraction and end-to-end automation for TSO 500 ctDNA library prep

Abstract Liquid biopsy such as plasma, has emerged as one of the most valuable sample types for profiling circulating tumor DNA (ctDNA) for screening, response to treatment, and monitoring relapse in oncology patients. Due to the diffuse nature of ctDNA however, both extraction and library preparation must be optimized for the efficient downstream next-generation sequencing (NGS) application. We initially addressed the optimization of quality and quantity of cfDNA recovery from healthy and various oncology patients by evaluating QIAsymphony DSP Circulating DNA Kit and Apostle MiniMax cfDNA isolation kits. Overall, the cfDNA yields from Apostle MiniMax kit were approximately 12X higher. We then optimized high-volume automated cfDNA extraction from 1ml, 3ml and 6ml plasma samples using the Biomek i7 for Apostle MiniMax kit. Colorectal, Gastric and Ovarian cancer gave the highest yield of cfDNA whereas, Esophageal, Lung and Kidney cancers were the lowest cfDNA yielding samples. We then analyzed ctDNA from plasma from healthy donors, colorectal, kidney, Breast, and endometrial cancer patients along with Oncospan and Seraseq ctDNA mutation mix (5-0.5% AF) to evaluate the mutation detection efficacy, repeatability, and reproducibility by targeted NGS using TSO 500ctDNA assay ay 10ng, 20ng, and 30ng inputs. For library preparation batches of 48 samples were prepared using a fully automated processes for the library prep, Hybridization capture and post capture enrichment on the Beckman i5 robot. Libraries were sequenced on NovaSeq 6000 at 600M Paired-end depth. This method demonstrates an overall concordance of inter-run and intra-run accuracy of variant detection to be 90% demonstrating that this method run as described produces highly repeatable and reproducible variant detection to sub-1% allele frequencies. The inter-run, lot-to-lot, and operator-to-operator variability was ≥99%. Down-sample analysis demonstrated high levels of assay performance with sample inputs of 30 ng with ≥96% sample passing Illumina cut-off metrics at 150M PE read depth as compared to Illumina recommended 420M PE reads. Taken together, we provide a high-volume automated solution for cfDNA extraction from plasma samples, additionally these data demonstrate a robust, reproducible, and highly accurate targeted DNA sequencing method that can be used for low allele frequency variant detection in cfDNA. Citation Format: Nripesh Prasad, Rachel Rock, Rebecca Beatty, Melanie Robinson, Dineen Wildman, Michael Sykes, Boris Umylny, Thomas Halsey. Optimization of high-volume cell free DNA extraction and end-to-end automation for TSO 500 ctDNA library prep [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2298.

Abstract 5018: Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications

Abstract “For research use only. Not for diagnostic procedures.” Introduction: Liquid biopsy is a non-invasive alternative to tissue biopsies for cancer profiling using cell-free total nucleic acids (cfTNA; including cell free DNA (cfDNA) and cell free RNA (cfRNA)), the biological analytes derived from tumor cells floating in blood. However, it is crucial to optimize the whole blood fractionation workflows, since limited access to a suitable centrifuge at blood collection sites poses a challenge to this critical fractionation step, causing delays in processing the blood, leading to decreased sensitivity of variant detection and increased occurrence of false positives. Current study shows a systematic comparison of the effects of 8 different blood fractionation conditions for spin speeds, temperatures and duration on the levels of cfTNA derived from K2-EDTA-derived blood from multiple healthy and non-small cell lung cancer (NSCLC) donors, using next generation sequencing (NGS). Methods: CfTNA from K2-EDTA derived plasma was isolated using the MagMAXTM Cell-Free Total Nucleic Acid kit and the Genexus™ Cell free total nucleic acid kit, utilizing the KingFisher TM Flex and GenexusTMpurification systems. Yield and integrity of cfTNA isolated was evaluated by Qubit™, Agilent Tapestation and quantitative real time PCR (qRT-PCR). Furthermore, the impact of these test conditions on quality control specifications of DNA and RNA was assessed by performing sequencing on an Ion TorrentTM GenexusTM sequencing instrument using the AmpliSeqTM HD target amplification assay, that reports sequence variations in DNA for 42 cancer related genes and RNA in 7 cancer related genes. Results: Results showed comparable performance for all the 8 blood fractionation conditions tested, for yield and cfDNA profiles compared to control condition, indicating that there is no significant impact of these conditions on either yield or integrity of the cfDNA isolated. In addition, NGS data also showed that on target reads and median molecular coverage for DNA are comparable for all the conditions tested. However, sequencing data demonstrated that one test condition showed significantly better performance for RNA quality control specifications in terms of RNA mapped reads and molecular coverage for process controls used in the assay, compared to all other conditions tested. Conclusion: Data suggests that one of the whole blood fractionation conditions tested impact the cfRNA recovery from plasma without negatively affecting the yield, and integrity of isolated cfDNA and shows increased sensitivity for RNA process controls in NGS. Citation Format: MADHU JASTI, Sergio Hernandez, Nick Siepert, Lokesh Kodandaramaswamy, Thilanka Jayaweera, Jeoffrey Schageman, Angie Cheng, Susanne Lopez, Miao Der Chen. Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5018.

Abstract 5030: Evaluation of different blood collection tubes for liquid biopsy NGS applications

Abstract Intro: Liquid biopsy is fast becoming a standard practice in oncology research due to recent advancements in Next Generation Sequencing (NGS). To detect genetic variants, whole blood samples are collected in blood collection tubes (BCTs) and fractionated to obtain plasma which is then used to isolate cell free nucleic acid (cfNA) for analysis. While the limited availability of cfNA is a challenge in liquid biopsy, storage condition and duration between blood collection and fractionation have a significant impact on the quantity and quality of the cfNA due to degradation and genomic DNA and RNA contamination. There are several BCTs available on the market offering stabilization of cfNA profiles to overcome the above challenges. Methods: Here we compare the NGS performance of cfNA derived from three commonly used BCTs on the market, BD Vacutainer™ K2EDTA Tubes, Streck™ Cell-Free DNA, and PAXgene® Blood ccfDNA tubes. Matched whole blood samples collected in the above-mentioned tube types were obtained from commercial vendors which were derived from 38 healthy donors as well as 6 cancer research samples. Whole blood samples were fractionated as per manufacturer recommendations and cfNA was isolated and quantified using Genexus™ Cell-Free Total Nucleic Acid Purification Kit on Genexus™ purification system. Isolated cfNA was then sequenced using Ion Ampliseq™ HD target amplification assay on Genexus™ Integrated sequencer. Results: Data shows that K2EDTA collected samples contain slightly higher amounts of cfNA compared to PAXgene® and Streck™ samples. When the cfDNA profiles were analyzed by Agilent™ BioAnalyzer™, K2EDTA derived cfDNA showed varying degrees of genomic DNA contamination which can inflate the cfDNA concentration. However, both Streck™ and PAXgene® derived cfDNA showed little to no detectable genomic DNA contamination. As a result, a higher percentage of PAXgene® and Streck™ samples did not meet the minimum input requirement (5ng) for the sequencing assay. Sequencing data for DNA showed that K2EDTA samples had higher total reads and median molecular coverage compared to the other two BCT types. However, all samples had passing QC metrics, and mean read length, on target reads, and uniformity were comparable between the three tube types. When DNA variant detection was evaluated using cancer research samples, no significant difference was observed in performance between K2EDTA and PAXgene® samples. Interestingly, sequencing data for RNA shows that PAXgene® samples had slightly higher mapped reads on average, and significantly higher internal process control coverage compared to both K2EDTA and Streck™ samples. Whereas Streck™ samples had significantly lower internal process control coverage. Conclusion: In summary, this study demonstrates the advantages and disadvantages of using different BCTs for liquid biopsy applications, in terms of cfNA yields, isolated DNA profiles, and sequencing performance. Citation Format: Nicholas Siepert, Emilia Ostrowska, Luming Qu He, Madhuri Jasti, Kris Lea, Thilanka Jayaweera, Angie Cheng. Evaluation of different blood collection tubes for liquid biopsy NGS applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5030.

Abstract 5054: Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications

Abstract Formalin-fixed paraffin-embedded (FFPE) biopsies are highly valuable and widely used tissue specimens for clinical diagnostics. However, obtaining sufficient and high-quality nucleic acid material from limited FFPE samples presents a challenge for downstream molecular analysis, such as next-generation sequencing (NGS). We present an optimized sequential extraction method that generates high-quality DNA and RNA from a single set of input tissues that is automatable and operation-friendly. This workflow performs well with reduced FFPE tissue input and efficiently supports various high-throughput clinical NGS applications. The DNA/RNA yield, quality, purity, and impacts on NGS assay performances were on par or better than an existing validated comparator extraction method. With comparable FFPE input, the new method demonstrated a superior extraction performance with significantly higher yield, quality, and purity. For DNA, NGS libraries were made with two different library preparation methods: a TA-ligation-based method and a single-strand-based method, followed by hybrid capturing and sequencing. The DNA from the new extraction method demonstrated a superior library conversion rate and improved target enrichment uniformity with both chemistries. This provides the potential of reduced input requirements, allowing very limited tissue, such as fine needle aspirate or core needle biopsy, for clinical NGS testing. The sequencing results were highly concordant between the existing and new extraction methods when the extracted DNA was subjected to a validated comprehensive genomic profiling (CGP) clinical test, demonstrating the quality and robustness of the extraction. For RNA, NGS libraries were made following the validated CGP clinical test with hybrid capturing, and the results were comparable between the two extraction methods. In conclusion, an FFPE extraction method was optimized to allow more clinical biopsy samples to be tested with different NGS workflows, providing a better diagnostic value for patient care. Citation Format: Rebecca Hong, Jieying Chu, Steven Hsiao, Daniel Whang, Steven P. Rivera, Heng Xie, Jiannan Guo. Optimization and evaluation of an FFPE dual extraction protocol for next-generation sequencing applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5054.

Abstract 3489: MetFlow: Automated nextflow pipeline for Ion AmpliSeq methylation assay designs

Abstract Introduction: DNA methylation plays a crucial role in development and progression of cancer. Identification of DNA methylation profiles in cancer is indispensable for early detection. To address this research need, Ion AmpliSeqTM offers targeted next-generation sequencing (NGS) methylation community amplicon panels for DNA methylation profiling in cancer-associated genes. However, the process of designing custom methylation amplicon panels using the Ion AmpliSeq primer design pipeline is intricate, labor-intensive, and time-consuming. Here, we developed a one-step, fully automated, faster, and reproducible nextflow-based primer design pipeline (MetFlow) for development of custom Ion AmpliSeqTM methylation panels. MetFlow streamlines the development of custom methylation panels for cancer-related genes, and thus significantly reduces the time and effort traditionally required for amplicon design. Methods: The fully automated MetFlow pipeline is implemented using Nextflow. MetFlow necessitate an input configuration file for the execution of methylation Ion AmpliSeqTM designs. The configuration file specifies the essential parameters, including target CpG input, reference genome, pool number, and amplicon lengths, which are required for designing the amplicons. The MetFlow pipeline designs candidate amplicons for CpG targets on both Watson and Crick strands, facilitating the subsequent creation of files required for the generation of a methylation panel. Results: The fully automated MetFlow pipeline has been used for designing the customized Ion AmpliSeqTM methylation amplicon panel(s) for cancer and age-related genes. The amplicons lengths in these AmpliSeqTM methylation panels were ranged from 125 bp to 175 bp for formalin-fixed, paraffin-embedded (FFPE) research samples. These customized methylation panels were run on the Ion GeneStudioTM S5 sequencer followed by performance analysis using Ion TorrentTM Suite. The published data for these custom amplicon panels suggest that the Ion AmpliSeqTM has sufficient read depth and mapping efficiency, higher sensitivity and accuracy for methylation measurement, and strong correlation (r=0.993) for methylation profiling among sample replicates. Conclusion: We present MetFlow, an advanced automated primer design pipeline for designing the custom Ion AmpliSeqTM methylation amplicon panels tailored to targeted NGS applications in cancer research. The custom designed Ion AmpliSeqTM methylation panels exhibited sufficient read depth, higher sensitivity and accuracy, and reproducibility. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Renesh Bedre, Drishti Kaul, Anupma Sharma, Andrew Hatch, Loni Pickle, Na Li, Fiona Hyland. MetFlow: Automated nextflow pipeline for Ion AmpliSeq methylation assay designs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3489.

Glanzmann Thrombasthenia 10 Years Later: Progress Made and Future Directions.

Glanzmann thrombasthenia (GT) is the most common inherited platelet disorder (IPD) with mucocutaneous bleeding and a failure of platelets to aggregate when stimulated. The molecular cause is insufficient or defective αIIbβ3, an integrin encoded by the ITGA2B and ITGB3 genes. On activation αIIbβ3 undergoes conformational changes and binds fibrinogen (Fg) and other proteins to join platelets in the aggregate. The application of next-generation sequencing (NGS) to patients with IPDs has accelerated genotyping for GT; progress accompanied by improved mutation curation. The evaluation by NGS of variants in other hemostasis and vascular genes is a major step toward understanding why bleeding varies so much between patients. The recently discovered role for glycoprotein VI in thrombus formation, through its binding to fibrin and surface-bound Fg, may offer a mechanosensitive back-up for αIIbβ3, especially at sites of inflammation. The setting up of national networks for IPDs and GT is improving patient care. Hematopoietic stem cell therapy provides a long-term cure for severe cases; however, prophylaxis by monoclonal antibodies designed to accelerate fibrin formation at injured sites in the vasculature is a promising development. Gene therapy using lentil-virus vectors remains a future option with CRISPR/Cas9 technologies offering a promising alternative route.

Application of metagenomic next-generation sequencing in the clinical diagnosis of infectious diseases after allo-HSCT: a single-center analysis

BackgroundWe investigated the value of metagenomic next-generation sequencing (mNGS) in diagnosing infectious diseases in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT).MethodsFifty-four patients who had fever following allo-HSCT from October 2019 to February 2022 were enrolled. Conventional microbiological tests (CMTs) and mNGS, along with imaging and clinical manifestations, were used to diagnose infection following allo-HSCT. The clinical diagnostic value of mNGS was evaluated.ResultsA total of 61 mNGS tests were performed, resulting in the diagnosis of 46 cases of infectious diseases. Among these cases, there were 22 cases of viral infection, 13 cases of fungal infection, and 11 cases of bacterial infection. Moreover, 27 cases (58.7%) were classified as bloodstream infections, 15 (32.6%) as respiratory infections, 2 (4.3%) as digestive system infections, and 2 (4.3%) as central nervous system infections. Additionally, there were 8 cases with non-infectious diseases (8/54, 14.81%), including 2 cases of interstitial pneumonia, 2 cases of bronchiolitis obliterans, 2 cases of engraftment syndrome, and 2 cases of acute graft-versus-host disease. The positive detection rates of mNGS and CMT were 88.9% and 33.3%, respectively, with significant differences (P < 0.001). The sensitivity of mNGS was 97.82%, the specificity was 25%, the positive predictive value was 93.75%, and the negative predictive value was 50%. Following treatment, 51 patients showed improvement, and 3 cases succumbed to multidrug-resistant bacterial infections.ConclusionsmNGS plays an important role in the early clinical diagnosis of infectious diseases after allo-HSCT, which is not affected by immunosuppression status, empiric antibiotic therapy, and multi-microbial mixed infection.

Application of Metagenomic Next-Generation Sequencing in Suspected Spinal Infectious Diseases

ObjectiveThis study aimed to explore the clinical efficacy of metagenomic next-generation sequencing (mNGS) in diagnosing and treating suspected spinal infectious diseases. MethodsBetween October 2022 to December 2023, a retrospective analysis was performed on patient records within the Department of Spinal Surgery at Guilin People's Hospital. The analysis included comprehensive data on patients with presumed spinal infectious diseases, incorporating results from mNGS tests conducted externally, conventional pathogen detection results, laboratory examination results, and imaging findings. The study aimed to assess the applicability of mNGS in the context of suspected spinal infectious lesions. ResultsTwenty-seven patients were included in the final analysis. Pathogenic microorganisms were identified in 23 cases. The included cases encompassed one case of tuberculous spondylitis, one case of fungal infection, three cases of Brucella spondylitis, three cases of viral infection, nine cases of bacterial infection, and six cases of mixed infections. Pathogenic microorganisms remained elusive in four cases. The application of the mNGS method demonstrated a significantly elevated positive detection rate compared to conventional methods (85.19% vs. 48.15%, P < 0.05). Moreover, the mNGS method detected a greater variety of pathogen species than traditional methods (Z = 10.69, P < 0.05). Additionally, the mNGS method exhibited a shorter detection time. ConclusionmNGS demonstrated significantly higher detection rates for bacterial, fungal, viral, and mixed infections in cases of suspected spinal infectious diseases. The clinical implementation of mNGS could further enhance the efficiency of diagnosing and treating suspected spinal infectious diseases.

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

Abstract. Jasim JHM, Othman AS, Nordin FA, Talkah NSM. 2024. High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing. Biodiversitas 25: 654-663. Advancements in genomic research have spurred a growing interest in extracting high-quality genomic DNA, particularly from intricate plant species like the Yellow Spathoglottis Blume Complex. The preparation of genomic DNA high-quality from a plant sample is a crucial step for genomic and genetic analysis studies. A variety of genomic DNA extraction methods such as the traditional Hexadecyltrimethylammonium Bromide (CTAB) method and commercially available kits have been reported. Even so, they are either low purity and yield or costly. We develop a good-quality genomic DNA extraction method from fresh and dried leaves of the Yellow Spathoglottis Blume complex in Peninsular Malaysia, specifically tailored for Next-Generation Sequencing (NGS) applications. Three DNA extraction methods were compared traditional CTAB, modified CTAB, and the DNeasy Qiagen plant mini kit methods. The yield and quality of extracted DNA were assessed by PCR amplification using nuclear ETS and ITS genes performed to evaluate the success of DNA extraction. Additionally, the suitability of the modified CTAB method for NGS library preparation and sequencing was tested. It demonstrated better PCR amplification results, yielding high-concentration bands for both ETS and ITS primers. Furthermore, the modified CTAB method proved suitable for high molecular weight DNA extraction, without contamination. The extracted DNA from Spathoglottis aurea exhibited high molecular weight and passed the quality control assessment for NGS library preparation. Sequencing results demonstrated an average quality score of 36.0, with an accurate base call rate of approximately 99.9%. The developed method enables reliable genomic analysis and sequencing of the Yellow Spathoglottis Blume complex, contributing to further research and understanding of this species group.

Clinical application of next generation sequencing for Mendelian disease diagnosis in the Iranian population

Next-generation sequencing (NGS) has been proven to be one of the most powerful diagnostic tools for rare Mendelian disorders. Several studies on the clinical application of NGS in unselected cohorts of Middle Eastern patients have reported a high diagnostic yield of up to 48%, correlated with a high level of consanguinity in these populations. We evaluated the diagnostic utility of NGS-based testing across different clinical indications in 1436 patients from Iran, representing the first study of its kind in this highly consanguineous population. A total of 1075 exome sequencing and 361 targeted gene panel sequencing were performed over 8 years at a single clinical genetics laboratory, with the majority of cases tested as proband-only (91.6%). The overall diagnostic rate was 46.7%, ranging from 24% in patients with an abnormality of prenatal development to over 67% in patients with an abnormality of the skin. We identified 660 pathogenic or likely pathogenic variants, including 241 novel variants, associated with over 342 known genetic conditions. The highly consanguineous nature of this cohort led to the diagnosis of autosomal recessive disorders in the majority of patients (79.1%) and allowed us to determine the shared carrier status of couples for suspected recessive phenotypes in their deceased child(ren) when direct testing was not possible. We also highlight the observations of recessive inheritance of genes previously associated only with dominant disorders and provide an expanded genotype–phenotype spectrum for multiple less-characterized genes. We present the largest mutational spectrum of known Mendelian disease, including possible founder variants, throughout the Iranian population, which can serve as a unique resource for clinical genomic studies locally and beyond.

Enrichment of Immune Dysregulation Disorders in Adult Patients with Human Inborn Errors of Immunity

Human inborn errors of immunity (IEI) comprise a group of diseases resulting from molecular variants that compromise innate and adaptive immunity. Clinical features of IEI patients are dominated by susceptibility to a spectrum of infectious diseases, as well as autoimmune, autoinflammatory, allergic, and malignant phenotypes that usually appear in childhood, which is when the diagnosis is typically made. However, some IEI patients are identified in adulthood due to symptomatic delay of the disease or other reasons that prevent the request for a molecular study. The application of next-generation sequencing (NGS) as a diagnostic technique has given rise to an ever-increasing identification of IEI-monogenic causes, thus improving the diagnostic yield and facilitating the possibility of personalized treatment. This work was a retrospective study of 173 adults with IEI suspicion that were sequenced between 2005 and 2023. Sanger, targeted gene-panel, and whole exome sequencing were used for molecular diagnosis. Disease-causing variants were identified in 44 of 173 (25.43%) patients. The clinical phenotype of these 44 patients was mostly related to infection susceptibility (63.64%). An enrichment of immune dysregulation diseases was found when cohorts with molecular diagnosis were compared to those without. Immune dysregulation disorders, group 4 from the International Union of Immunological Societies Expert Committee (IUIS), were the most prevalent among these adult patients. Immune dysregulation as a new item in the Jeffrey Model Foundation warning signs for adults significantly increases the sensitivity for the identification of patients with an IEI-producing molecular defect.