Next-generation Sequencing Sequencing Research Articles

Assessment of the pathogen genomics landscape highlights disparities and challenges for effective AMR Surveillance and outbreak response in the East African community

The East African Community (EAC) grapples with many challenges in tackling infectious disease threats and antimicrobial resistance (AMR), underscoring the importance of regional and robust pathogen genomics capacities. However, a significant disparity exists among EAC Partner States in harnessing bacterial pathogen sequencing and data analysis capabilities for effective AMR surveillance and outbreak response. This study assesses the current landscape and challenges associated with pathogen next-generation sequencing (NGS) within EAC, explicitly focusing on World Health Organization (WHO) AMR-priority pathogens. The assessment adopts a comprehensive approach, integrating a questionnaire-based survey amongst National Public Health Laboratories (NPHLs) with an analysis of publicly available metadata on bacterial pathogens isolated in the EAC countries. In addition to the heavy reliance on third-party organizations for bacterial NGS, the findings reveal a significant disparity among EAC member States in leveraging bacterial pathogen sequencing and data analysis. Approximately 97% (n = 4,462) of publicly available high-quality bacterial genome assemblies of samples collected in the EAC were processed and analyzed by external organizations, mainly in Europe and North America. Tanzania led in-country sequencing efforts, followed by Kenya and Uganda. The other EAC countries had no publicly available samples or had all their samples sequenced and analyzed outside the region. Insufficient local NGS sequencing facilities, limited bioinformatics expertise, lack of adequate computing resources, and inadequate data-sharing mechanisms are among the most pressing challenges that hinder the EAC’s NPHLs from effectively leveraging pathogen genomics data. These insights emphasized the need to strengthen microbial pathogen sequencing and data analysis capabilities within the EAC to empower these laboratories to conduct pathogen sequencing and data analysis independently. Substantial investments in equipment, technology, and capacity-building initiatives are crucial for supporting regional preparedness against infectious disease outbreaks and mitigating the impact of AMR burden. In addition, collaborative efforts should be developed to narrow the gap, remedy regional imbalances, and harmonize NGS data standards. Supporting regional collaboration, strengthening in-country genomics capabilities, and investing in long-term training programs will ultimately improve pathogen data generation and foster a robust NGS-driven AMR surveillance and outbreak response in the EAC, thereby supporting global health initiatives.

Bacterial communities inhabiting the ascomata of the ectomycorrhizal summer truffle (Tuber aestivum)

This paper continues the studies on the role of bacteria in the environment of summer truffle (Tuber aestivum), focusing on ascomata at three sites in the Nida Basin (southern Poland). Bacterial taxa and the number of OTU sequences identified were determined based on analysis of peridium and gleba collected in 2017 and 2018. The aim of the presented work is to learn the structure of bacterial communities associated with the ascomata of the summer truffle T. aestivum forming in specific stands in south Poland and fine roots of surrounding trees. Sanger DNA sequencing, and next-generation sequencing (NGS) were used. NGS sequencing platforms short-read (300 bases) was selected, using a MiSeq Illumina. The Kruskall-Wallis test was used for alpha diversity analysis and the PERMANOVA test for beta diversity. Alpha and beta biodiversity analyzes were performed using various indices (Chao1, Simpson, Shannon, Faith's phylogenetic, observed OTUs, Heip's evenness measure - alpha diversity; UniFrac, Jaccard, Bray–Curtis - beta diversity).The Sanger method identified six bacterial taxa that were dominant in the community: Bacillus (86.7 %), Pseudomonas (51.5 %), Stenotrophomonas (15.5 %), Paenibacillus (7.9 %), Rhizobium (7.3 %), and Chryseobacterium (8.5 %). In contrast, NGS results showed the dominance (at OTUs >10,000) of taxa: Bradyrhizobiaceae, Sphingobacteriaceae, Micrococcaceae, Yersiniaceae, Sphingomonadaceae, Rhizobiaceae, Flavobacteriaceae, and Comamonadaceae. The PERMANOVA test for the Bray-Curtis and Jaccard indices showed dissimilarities between samples from two of the sites with respect to beta diversity. Significant differences were found in the bacterial communities colonizing the ascomata of T. aestivum and in the root communities with T. aestivum ectomycorrhizae the purpose of this study. The results obtained may be helpful in selecting and monitoring sites suitable for the formation of ascomata of T. aestivum.

Impact of electrolyzed water treatment on bacterial communities in food washing processes

Electrolyzed water is expected to have a sterilizing effect on bacteria and viruses, which makes it useful for hygiene management, such as cleaning dishes or medical equipment. Electrolyzed water treatment may affect the cellular structure of microbes, leading to DNA leakage or degradation. Here, we conducted a next-generation sequencing (NGS) analysis of bacterial diversity in washing solution to elucidate the impact of cleaning food surfaces with electrolyzed water. Modified primers were used to evaluate the differences between the existing primers used to obtain NGS sequences and through statistical analysis. The NGS reads obtained using the modified primers tended to reveal more Enterobacteriaceae sequences, and fewer mitochondrial sequences than did those obtained with conventional primers. An UniFrac distance analysis and Nonmetric multidimensional scaling (NMDS) revealed that the results obtained with the modified primer set made the differences between samples more distinct. Real-time polymerase chain reaction (PCR) using the template DNA obtained from an electrolyzed water or sodium hypochlorite solution wash revealed that some of the DNA could not be amplified using PCR of the 16S rRNA genes suggesting that the DNA fragmentation had occurred due to the electrolyzed water treatment. Our results revealed that the bacterial species removed from the food surfaces varied depending on the washing treatment. Electrolyzed water treatment may be advantageous for removing Enterobacteriaceae from leafy vegetables.

Abstract 2949: A flexible and efficient approach for single-cell CRISPR perturbation screens combined with targeted RNA-seq expression analysis

Abstract This study presents a novel approach for performing 10X platform-based targeted single-cell RNA-seq analysis in combination with CRISPR perturbation screens. For the analysis, we constructed a custom sgRNA library focused on a small subset of genes associated with the TNFα response (i.e., NFκB pathway). We compared analysis of two targeted gene expression panels—the first, targeting all protein-coding genes (genome-wide) and the other, targeting a selected set of genes representing key hubs in signaling pathway modules. Narrowing down the analyzed gene set enhanced the efficiency of single-cell RNA-seq by reducing the associated next-generation sequencing (NGS) depth required for readout, and significantly streamlining data analysis. Comparing the analysis of the expression panels targeting all protein-coding genes with the smaller pathway-targeted set, we were able to assess the general flexibility of the novel approach for single-cell CRISPR perturbation screens. We evaluated the specific tradeoffs with regard to the size of the CRISPR library, the size of the panel of genes targeted for expression analysis, and NGS sequencing depth. This flexible, targeted approach not only refines the precision of RNA-seq but also provides a more cost-effective solution for comprehensive genomic analyses. Citation Format: Donato Tedesco, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Nadya Isachenko, Paul Diehl, Alex Chenchik. A flexible and efficient approach for single-cell CRISPR perturbation screens combined with targeted RNA-seq expression analysis [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 2949.

Identification of gene variation feature for targeted therapy of non-small cell lung cancer through combined method of DNA and RNA sequencing

Next generation sequencing (NGS) is typically used to reveal tumor gene variation feature for targeted therapy of various types of human cancers, including non-small cell lung cancer (NSCLC). Here, we report the role and potential applicable value of combining DNA and RNA sequencing in gene variation detection in NSCLC. 386 NSCLC patients with stage II-IV were enrolled and detected using NGS sequencing of DNA and RNA panels that covered all well-documented target driver genes from the Chinese Society of Clinical Oncology (CSCO). The rate of epidermal growth factor receptor (EGFR) single nucleotide variation (SNV)/indel, mesenchymal-epithelial transition factor (MET) copy number variation (CNV) and anaplastic lymphoma kinase (ALK) fusion were 52.1%, 4.1% and 6.0% in the NSCLC cohort. The landscapes of SNV/indel, CNV and gene fusion in the cohort were depicted as well. Meanwhile, we assessed detection efficacy of DNA and RNA sequencing in gene fusion. Detected number and types of gene fusion using the RNA sequencing were better than those using the DNA sequencing. Gene fusion with intergenic region was only detected by DNA sequencing and MET exon 14 skipping (METΔex14) was more easily identified by RNA sequencing. Finally, we investigated clinical correlations of SNV/indel/CNV/fusion with clinicopathologic features in the NSCLC cohort. Taken together, RNA sequencing significantly complements deficiency of DNA sequencing for gene fusion, which cooperatively presents comprehensive and reliable gene variation features and facilitate the identification of potential drug targets for NSCLC patients.

Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants

BackgroundKrüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants.ResultsWe included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected.ConclusionsHigh-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

Author Correction: Genetic variants identified in novel candidate genes for anorexia nervosa and analysis of molecular pathways for diagnostic applications.

Correction to: Eur Rev Med Pharmacol Sci 2023; 27 (6 Suppl): 77-88-DOI: 10.26355/eurrev_202312_34692 After publication and following some post-publication concerns, the authors have applied the following corrections to the galley proof. The conflict of interest section has been amended as follows: K. Donato is employee at MAGI EUREGIO and MAGISNAT. G. Marceddu is employee at MAGI EUREGIO. M. Bertelli is president of MAGI EUREGIO, MAGISNAT, and MAGI's LAB. M.C. Medori, A. Macchia, S. Cecchin, C. Micheletti, K. Dhuli, G. Madeo, G. Bonetti are employees at MAGI's LAB. M. Bertelli, M.R. Ceccarini, and P. Chiurazzi are patent inventors (US20220362260A11). M. Bertelli, P.E. Maltese, G. Marceddu, and S. Cecchin are patent inventors (US20230173003A1). M. Bertelli, K. Dhuli, and P.E. Maltese are patent inventors (WO2022079498A1). M. Bertelli, K. Donato, M.C. Medori, M.R. Ceccarini, T. Beccari, P. Chiurazzi, C. Micheletti, K. Dhuli, G. Bonetti, G. Marceddu are patent applicants (Application Number: 18/466.879). The remaining authors have no conflict of interest to disclose. Since the current study shares the same NGS panel for the genetic analysis as the study cited in Ref. 5 (Ceccarini MR, Precone V, Manara E, Paolacci S, Maltese PE, Benfatti V, Dhuli K, Donato K, Guerri G, Marceddu G, Chiurazzi P, Dalla Ragione L, Beccari T, Bertelli M. A next generation sequencing gene panel for use in the diagnosis of anorexia nervosa. Eat Weight Disord 2022; 27: 1869-1880), the authors amend the following sentence: "A subset comprising 163 genes from a dedicated Next-Generation Sequencing (NGS) panel was analyzed5" in "A subset comprising 163 genes from a dedicated Next-Generation Sequencing (NGS) panel, previously used in the study by Ceccarini et al5, was analyzed". The authors clarify that the analyzed patients of the two articles are completely independent. To clarify the data reported in Table II, the authors amend the following sentence: "Genetic variants identified in the AN population are reported in Table II." In "The genomic sequencing NGS was performed in all 135 patients recruited in the study. After obtaining the raw data, based on the ACMG guidelines (https://www.acmg.net/ACMG/Medical-Genetics-Practice-Resources/Practice-Guidelines.aspx), the results were filtered, and Table II reports the variants considered Pathogenic (P), likely pathogenic (LP), and Variable with Uncertain Significance (VUS), 61 patients in total". Consequently, to improve clarity, the legend of Table II has been amended as follows: Genetic variants identified in 61 patients out of the total 135 patients analyzed by NGS. There are amendments to this paper. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/34692.

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.

Molecular biology techniques for assessing the loss of HLA heterozygosity after allogeneic hematopoietic stem cell transplantation in children with acute leukemia

According to several observations, up to a third of post-transplant relapses in childhood acute leukemia are associated with the loss of heterozygosity of the major histocompatibility complex (HLA). Furthermore, the inefficacy of the graft-versus-leukemia reaction, as evidenced by the lack of therapeutic effect from the infusion of donor lymphocytes, indicates the need for timely detection of this marker to change the treatment strategy in the post-transplant period. To detect the loss of HLA heterozygosity, the method using the commercial KMR-HLA system and analysis using next-generation sequencing (NGS), as well as the method based on the analysis of highly polymorphic STR and VNTR markers located in the HLA loci region on the short arm of chromosome 6, are widely used. The primary objective of our study was to compare the informativeness of these approaches in diagnosing HLA heterozygosity loss in children during the post-transplant period. The obtained data on the frequency of detecting HLA heterozygosity loss were comparable to the literature data and constituted 23 % of cases of post-transplant relapse of B-cell acute lymphoblastic leukemia, 33 % of cases of T-cell acute lymphoblastic leukemia, and 23% of cases of acute myeloid leukemia. We also demonstrated that the method based on STR marker analysis has sensitivity comparable to allele-specific PCR and NGS sequencing methods. Meanwhile, preliminary sorting of the blast population increases the sensitivity of STR analysis and can be recommended in routine practice.

Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing.

Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

Clinical and Molecular Characterization of Triple-Negative Essential Thrombocythemia: Data from the Prospective Spanish Registry of Essential Thrombocythemia

Introduction Triple-negative Essential Thrombocythemia (TN-ET) accounts for up to 10-20% of patients with a histological diagnosis of ET. Despite being considered an indolent disease, it remains a diagnostic and therapeutic challenge as it can be confused with non-clonal thrombocytosis and its prognostic factors and management are not standardized. Objectives The aim of this study was to evaluate the utility of a Next Generation Sequencing (NGS) panel targeting recurrently mutated genes in myeloid pathology for detecting genetic variants in patients diagnosed with TN-ET and to assess the significance of these variants in the risk of thrombosis and hemorrhage, progression to Acute Leukemia (AL) or Myelofibrosis (MF), and overall survival. Materials and Methods Data from 151 TN-ET patients with confirmed biopsy and NGS sequencing from the Spanish Registry of Essential Thrombocythemia of the Spanish Group of Philadelphia-Negative Myeloproliferative Neoplasms was obtained. This registry includes information on diagnosis, molecular biology, clinical characteristics, and molecular biology. In addition, 39 patients whose molecular diagnosis was performed at the Molecular Biology Laboratory of the Hematology Department at Hospital Universitario 12 de Octubre, Madrid, Spain were included. This last panel included the genes: ANKRD26, ASXL1, ATG2B, BCOR, BCORL1, CALR, CBL, CEBPA, CSF3R, CUX1, DDX41, DNMT3A, EGLN1, EPAS1, EPOR, ETNK1, ETV6, EZH2, FLT3, GATA2, GNAS, GNB1, GSKIP, IDH1, IDH2, JAK2, KDM6A, KIT, KMT2A, KRAS, MBD4, MPL, NF1, NFE2, NPM1, NRAS, PHF6, PPM1D, PRPF8, PTPN11, RAD21, RIT1, RRAS, RUNX1, SAMD9, SAMD9L, SETBP1, SF3A1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG2, TERC, TERT, TET2, THPO, TP53, U2AF1, VHL, WT1, ZRSR2. Statistical analyses were performed using SPSS v.29.0. Results A canonical mutation was detected in 20 out of the 190 studied patients, ( JAK2 V617F n=13, CALR n=3 and MPL n=4) being discarded from subsequent studies. Among the other 170 samples, 206 genetic variants were detected in a total of 86 patients (median 2, range 1-9). A total of 39 pathogenic (P) or probably pathogenic (PP) variants were found in 28 patients, whereas 59 uncertain significance (US) and 108 benign (B) variants were found in 44 and 54 patients, respectively. The distribution of genes in which P/PP/US variants were detected is shown in Figure 1. Patients with P /PP mutations had a statistically higher age compared to the remaining (50 vs. 64.8 years; p<0.001). The prevalence of males was higher in the group where P/PP mutations were detected (57.14% vs. 28.87%; p=0.004). No differences were found regarding cardiovascular comorbidity or symptomatology at diagnosis, but the P/PP group presented a higher frequency of thrombosis prior to diagnosis (28.57% vs. 5.64%; p=0.001). There were no differences in hemoglobin, leukocyte, or platelet counts at diagnosis according to the presence of P/PP variants. The presence of P/PP mutations was associated with a higher risk of progression to AL and a lower overall survival. A higher risk of thrombosis or progression to MF was not found (Figure 2). No differences were found in the risk of thrombosis, transformation, or overall survival between patients with or without the presence of US, PB or B variants. Conclusions The use of NGS technology is useful for the diagnosis and prognostication of TN-ET. The detection of P/PP mutations in patients diagnosed with TN-ET is associated with a higher risk of transformation to AL and lower overall survival, but not with higher risk of thrombosis or progression to MF. It is necessary to evaluate the individual risk of each of these mutations and the potential prognostic role of variants of US.

Mutations in Histone Lysine Methyltransferase Genes Are Associated with Autoimmune Cytopenias

Introduction Epigenetic mechanisms are important in regulation of gene expression by modifying chromatin structure to facilitate DNA accessibility (Lau et al Nat Immunol. 2018). DNA methylation and histone modifications are critical for the pathogenesis of hematologic malignancies and autoimmune diseases (Surace et al Front Immunol. 2019) Alterations in enzymes involved in histone modification are associated with immunodeficiencies, including Kabuki syndrome (KS), a rare genetic disorder associated with autoimmune cytopenias, such as immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA) (Margot et al J Am Coll Med Genet 2020) caused by loss-of-function KMT2D and KDM6A mutations. Somatic mutations in the KMT2D gene have also been observed in patients with Cold Agglutinin Disease. (Małecka et al Br J Haematol. 2018). Aims: The aims of this study are: 1) to identify the frequency of mutations in histone methyltransferase genes (MT) KMT2D, KMT2A, KMT2C and KDM6A in patients with autoimmune cytopenias, and 2) to compare clinical, laboratory and immunological characteristics, and response to therapy in patients with or without mutation in MT and autoimmune cytopenias (AIC). Methods Data mining software from the electronic medical record was used to identify patients ≥ 18 years with a diagnosis of AIC, including ITP, warm and cold AIHA, autoimmune neutropenia, and Evans Syndrome (ES), who had Next Generation Sequencing (NGS) performed. Cytopenias due to malignancy, nutritional deficiency, medications, pregnancy, or infection were excluded. Patients were categorized into two groups based on the presence or absence of mutation in the MT genes (MT+, MT-) with variant allelic frequency (VAF) > 2%. Demographic, laboratory, and clinical characteristics, treatment outcomes, and NGS sequencing data were collected and compared between groups. Next, KMT2 mutations seen in patients with AIC were cross-referenced against the 1000 Genome Project (1000GP) population database to evaluate the frequency of polymorphisms in the general population (Auton et al Nature 2015). CADD in silico prediction algorithm was used to score the potential deleterious effect of MT mutations (Kirchner Nat Genet. 2014). Chi-Square test was used for categorical variables. Statistical analysis was completed in R software. Results Among 495 patients who underwent NGS, 79 (16%) had a mutation in MT genes. These mutations were present in 36% of patients with AIC as compared to 13% in those without AIC (p=0.0005). Among 55 patients with AIC, mutations in the MT genes were present in 20 patients (MT+), 35 patients did not have these mutations (MT-). KMT2C mutations were seen in 10 (50%) patients, KMT2D mutations in 9 patients (45%, one patient had two KMT2D mutations), 1 patient (5%) had both KMT2C & KMT2D variants (Figure 1). The median VAF was 39.5% (IQR, 15.45 - 49.80). The MT+ group had a significantly higher proportion of Hispanic (60% vs 31%) and non-Hispanic White patients (25% vs 14%), p=0.038, Table 1. The median age at diagnosis for the MT+ cohort was younger compared to MT- (45.5 vs 58 years old, respectively), though not statistically significant. AIC diagnoses in both groups were ITP (44%) wAIHA (22%) cAIHA (9%) Autoimmune Neutropenia (2%), and ES (24%). Compared to the MT- group, the MT+ group had lower IgG levels (p=0.0041) and absolute lymphocyte count (p=0.041). MT+ patients with AIHA were more likely to have positive Direct Coombs and complement involvement. No significant differences were found in median nadir hemoglobin and platelet counts, need for therapy, or median number of lines of therapy. Best overall response to therapy was similar in the two groups (93%), with more complete responses in MT- group (55% vs 33%) though not statistically significant. Next, we assessed KMT2 polymorphisms in the general population. Most of the polymorphisms were not present in the 1000GP database and none had a frequency > 0.001. The average Phred score was 19.8; 12/20 patients had Phred score > 20, suggesting that most mutations are in the top 1% of the deleterious variants. Conclusion We report for the first time that patients with autoimmune cytopenias have a high frequency of variants in MT genes. Median allelic frequency close to 50% suggests potential germline predisposition to immune dysregulation and autoimmunity however further studies are needed to better understand the impact of these observations.

The Prognostic Value of Common Cardiovascular Biomarkers in the Survival of Patients with DLBCL

Background Diffused large B-cell lymphoma (DLBCL) is a subgroup of highly heterogenous and aggressive hematological malignancy. IPI scoring tools have been used to predict survival in DLBCL patients retrospectively. Moreover, it is indicated that cardiovascular biomarkers can predict long-term survival in myelodysplastic syndrome. To structure a more accurate prognostic model directing survival, we constructed retrospective research in our clinical institution with common cardiovascular biomarkers, for instance, IL-6, Troponin I, BNP, hydroxybutyrate dehydrogenase and creatine phosphokinase. Besides, other risk factors of cardiovascular diseases such as lipids are also taken into consideration. Method We retrospectively researched a total of 284 patients who were first diagnosed with DLBCL from January 2011 to November 2022, among whom 205 patients were sequenced by next-generation sequencing (NGS) before receiving therapy. Characteristics of democracy and clinical data were collected and analyzed. The primary objective is to investigate the prognostic value of common cardiovascular biomarkers in the long-term survival of DLBCL patients. Results A total of 284 patients were enrolled with a median follow-up time of 21.8 months. The inclusive criteria are: patients who are newly diagnosed with DLBCL and clinical data of cardiovascular mark can be retrospect, moreover, to eliminate the influence of cardiac failure we excluded those whose ejection fraction was lower than 30%. There are 78 patients with high levels of BNP (HIGH-BNP group) and 119 patients with high levels of IL-6 (HIGH-IL6 group). In the HIGH-BNP group, overall survival is poorer than the control group (p<0.001, HR=3.07, 95%CI 1.97-4.78). Besides, in the HIGH-IL6 group, the tendency shows similitude, with a lower survival ratio (p=0.025, HR=2.65, 95%CI 1.30-5.40). Among 205 patients who received NGS sequencing, 2270 genes mutation have been detected. the most frequently detected genes are PIM1 (324, 14.3%), KMT2D (73, 3.2%), BCL6 (80, 3.5%), TP53 (63, 2.8%) and MYD88 (57, 2.5%). Conclusion Cardiovascular biomarkers can properly predict DLBCL patient survival.

Efficacy and Safety of Zanubrutinib-Containing Regimens in Patients with Secondary CNS Relapse in Diffuse Large B-Cell Lymphoma

Background: In the era of immune-targeted therapy represented by rituximab, despite improved remission and survival rates in patients with diffuse large B-cell lymphoma (DLBCL), central nervous system (CNS) relapse, occurring in some DLBCL patients after achieving remission, remains a clinical challenge. Zanubrutinib is one of a new generation of BTK inhibitors that have higher blood-brain barrier permeability compared to other BTK inhibitors. Research Design and Methods: We performed a multicenter retrospective study to evaluate the efficacy and safety of Zanubrutinib-containing regimens for salvage treatment in 28 relapsed DLBCL patients with CNS involvement treated in 6 centers from January 2020 to December 2021. Results: The IPI score and CNS-IPI score did not have good enough predictive significance for lack of consistency of the median time from the initial diagnosis to CNS recurrence. Of the 28 patients, 16 had targeted next-generation sequencing (NGS) data. NGS test was performed on their tissue samples that were obtained at initial diagnosis and/or at the timepoint of CNS relapse in DLBCL. 8 at the time of initial diagnosis and 10 at the time of recurrence, with 2 of them having undergone NGS sequencing at both initial diagnosis and recurrence. Median study follow-up from CNS relapse was 8.55 months, all patients were evaluated for response with 20/28 (71.4 %) showing a response: 11 CR, 9 PR, and 4 SD, 4 PD. The median PFS is 6.6 months, and the median OS has not yet been reached. Notably, MYD88, CD79B, or TP53 mutations tested at diagnosis in these patients were associated with significantly more favorable treatment response, while patients with a dual rearrangement of MYC and BCL2 and/or BCL6 genes did not get a treatment response to Zanubrutinib-containing regimens. It has well-tolerated toxicity with few adverse events requiring treatment discontinuation or dose reduction. Conclusion: Zanubrutinib-containing regimens extended PFS and OS in relapsed DLBCL patients with CNS involvement.

Analysis of Clinical Features, Prognostic Characteristics and Gene Mutations of the EB Subtype of Primary Myelodysplastic Syndrome Patients with Myelofibrosis

Objective ：This study aims to analyze the clinical features, prognostic characteristics and gene mutations of the primary myelodysplastic syndrome with myelofibrosis (MDS-MF) patients, to compare the differences and impact of MF on MDS-EB group and MDS-Non-EB group, ultimately to improve the cognition of MF on MDS-EB group. Methods ：From January 1, 2013 to February 1, 2020, 417 newly diagnosed primary MDS patients in the first affiliated hospital of Zhejiang University with bone marrow (BM) biopsy examination were included. They were divided into two groups (MDS-EB group and MDS-Non-EB group) according to 2016 WHO MDS classification.BM was reassessed on whether associated with fibrosis in grade MF0,1,2,3. (2005 European Myelofibrosis Network criteria). The clinical features, prognosis characteristics and gene alterations were analyzed between the MDS-EB group and MDS-Non-EB group retrospectively. Results ： ① MF was confirmed in 46.3％（193/417）cases of all the MDS patients, of which 66.3％（128/193）were MF-1 , 25.9％（50/193）were MF-2 and 7.8%(15/193) were MF-3 respectively. Compared with MDS-MF0 group, MDS-MF were significantly associated with worse OS(P=0.035) and worse PFS(p<0.001). ② There was 174 cases of MDS-EB and 243 cases of MDS-Non-EB. The proportion of MF in MDS-EB group was 54.0%(94/174) and significantly higher than in MDS-Non-EB group 40.7%（99/243）(p=0.010).The distribution of MF grade was also differently significant between the two groups(p=0.048). The clinical features analysis showed that there is higher level of C-reactive Protein (CRP), higher level of serum lactate dehydrogenase (LDH) and higher level of β2-microgram in the peripheral blood in the MF(MF1,2,3) group than in the MF0 group, regardless of whether it was in the EB group or the non-EB group. The difference was statistically significant (ALL p value<0.05). ③The impact of MF on MDS-EB patients and MDS-Non-EB patients has different significances concerning OS, PFS and t-AML. As far as the OS prognostic factor, no matter the comparison of MF0, 1,2,3 four groups, the comparison MF1,2,3 three groups, the comparison of MF0-1 with MF2-3, all showed that MF was an adverse prognostic factor in the EB-MF group (All p value <0.05), and it also showed the adverse impact related with the grade of MF, but none of any in the MDS-Non-EB group (All p value>0.05)(Figure1). Comes to the PFS, it showed the MF on MDS-Non-EB was an inferior factor when compared MF+(MF1,2,3) to MF-(MF0)(p=0.0097), but not between the comparison MF1,2,3 groups(p=0.96) and none of the MDS-EB groups（p=0.31 and 0.092 respectively）. The incidence of MF was related with a higher rate of t-AML not only in MDS-EB group but also in MDS-Non-EB group compared with MF0, no matter compared the MF+(MF1,2,3) with MF-(MF0) but also the comparison of MF0,1,2,3) (All P value < 0.001). ④ In the MDS-EB cohort, 66 patients in the MF-(MF-0) group completed Next-generation-sequencing (NGS) test, and 61 patients in the MF+ (MF1,2,3) group completed NGS testing. It seems that the incidence of gene mutation has no difference between the two groups. We conducted univariate COX regression analysis about the 30 genes (which were suggested in the NCCN guideline 2020) with OS, PFS and t-AML in the MDS-EB-MF cohort. The results showed that TP53, Nras, WT1 was related to the worse OS; U2AF1, Niras and TP53 was related to worse PFS and GATA2, IDH2, TP53, STAG2 was related to t-AML. Conclusion: MDS-MF has unique laboratory and clinical characteristics, act as an independent risk factor for shorter OS and worse PFS in the MDS-MF patients in our large cohort. In this study, we retrospectively analyzed 417 primary MDS cases in our hospital and analyzed the impact of MF on MDS-EB group and MDS-Non-EB group respectively. Our results showed the MF has a worse OS on the MDS-EB-MF group than the MDS-EB-MF0 group but no impact on MDS-Non-EB cohort. It was also consistent with the 5th edition of the WHO Classification of Haemato-lymphoid Tumors guidelines in which MDS-f was listed as a subtype concerning MDS with fibrosis concomitant with 5-19% blast cells in bone marrow or 2-19% blast cells in peripheral blood. The NGS sequencing showed TP53, Nras, WT1 was related to the worse OS; U2AF1, Niras and TP53 was related to worse PFS and GATA2, IDH2, TP53, STAG2 was related to t-AML.As far as we know, this is the first analysis of bone marrow fibrosis in MDS-EB patients. We need more research to help us understand the MDS-f subtype.

Exploratory Analyses of Lymph Node Tissue, Serum, and Peripheral Blood Mononuclear Cells Do Not Reveal Pathogen Signatures in Idiopathic Multicentric Castleman Disease

Background : Castleman disease (CD) encompasses a group of hematologic disorders that share characteristic histopathological features. Unicentric CD (UCD) involves a solitary enlarged lymph node with typically mild symptoms. Multicentric CD (MCD) involves generalized lymphadenopathy and often severe cytokine-driven multi-organ dysfunction. MCD is further subdivided into cases caused by Human Herpesvirus-8 (HHV-8-associated MCD) infection, POEMS syndrome (POEMS-associated MCD), and those with an idiopathic cause (iMCD). The underlying pathological mechanisms and etiologies of UCD and iMCD are not well understood. Given that a viral pathogen, HHV-8, causes HHV-8-associated MCD, we pursued multiple approaches to identify potential pathogens that may trigger iMCD and UCD. Methods: We used 2 distinct detection methods to identify potential pathogens in CD. PathoChIP is a microarray-based assay that uses ~60,000 DNA/RNA probes to detect 4,000+ pathogens associated with human diseases. Viral Track is a computational method that can be applied to unmapped next-generation sequencing (NGS) reads to search for the presence of viral RNA. Formalin-fixed paraffin-embedded (FFPE) lymph node tissue was analyzed by PathoChIP from iMCD (n=6), UCD (n=8), HHV8-associated MCD (n=1, HHV8-MCD), EBV-driven post-transplant lymphoproliferative disorder (n=1, EBV-PTLD), reactive samples without a clear etiology (n=1), and POEMS-associated MCD (n=2) patients. We also analyzed serum from iMCD patients (n=2) and healthy donors (n=8). A ‘positive’ probe for a given microorganism was defined as exceeding the 95 th percentile on a per-probe basis of the optical signal captured across all probes in the dataset. Data from single-cell RNA sequencing of cryopreserved peripheral blood mononuclear cells (iMCD, n=4; EBV, n=2; HHV8, n=1; healthy donor, n=2) and bulk RNA sequencing of FFPE lymph node tissue (iMCD, n=9; sentinel, n=7; systemic lupus erythematosus (SLE), n=3; Diffuse large B-cell lymphoma (DLBCL), n=3) were used as input into the Viral Track program. To detect a positive viral signal, standard thresholds were required: >50 uniquely mapped reads to a given virus, a read complexity threshold >1.2, and >10% coverage of the length of the respective viral genome. Results: After quality control, 49,657 probes from the PathoCHIP assay were mapped to 4,000+ known pathogens. As expected, the HHV-8 MCD and EBV-PTLD samples identified significantly more positive probes compared to the rest of the cohort for HHV-8 (65/259 vs 1/259) and EBV (38/235 vs 1/235), respectively. Importantly, no other microorganisms were detected in UCD or MCD lymph nodes or serum samples at a significantly increased rate than controls. For Viral Track, human endogenous retrovirus K113 and Escherichia phages were detected at low levels throughout all results. Excluding these viruses, positive controls including HHV-8-MCD, and EBV solely identified their respective predicted virally mapped NGS reads and no viral signature was detected in healthy donors. Importantly, there was no significant viral signature specific to CD in PBMCs or LN tissue. Interestingly, 1 iMCD sample did contain a significant number of NGS sequencing reads mapping to HHV5. These results were confirmed with clinical data reporting a positive IgG, negative PCR, and negative IgM test within 2 weeks of the biopsy date, suggesting that the HHV5 positive result was likely detecting a previous, now latent infection rather than a potential pathological driver of CD. Conclusions: Although there are limitations to this study, including small cohorts and viral mRNA single-cell capture limitations, we were able to use 2 orthogonal pathogen detection methods on multiple tissue types from iMCD patients. Taken together, our results suggest that an active infectious agent is unlikely to be a major contributor or pathological mechanism driving UCD or iMCD. The continued search for potential causative microorganisms in iMCD patients should continue, but our results suggest that alternative hypotheses related to the pathology and etiology of iMCD and UCD should be prioritized.

B-253 Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing

Abstract Background High-resolution human leukocyte antigen (HLA) typing is a crucial process in transplant matching. Sequencing-based typing (SBT) has been the gold standard method used in HLA-based clinical applications. Recently, next-generation sequencing (NGS) has been adopted in clinical laboratories for HLA typing. NGS HLA typing assays cover whole or near-whole target HLA genes, allowing the detection of genetic variants outside of key exons. Multiplex amplicon-based NGS is an efficient high-resolution HLA typing method with minimal ambiguity and a high throughput. Here, we report the performance and validation of NGS for HLA typing and compare the results obtained using NGS with those of SBT to identify any discrepancies between the 2 methods. Methods A total of 237 samples from 2019 to July 2022 were collected from Chang Gung Memorial Hospital, including 42 samples previously subjected to SBT sequencing for 4-digit HLA analysis. NGS was performed for 5 whole genes (HLA-A/B/C/DQA1/DPA1) and 3 near-whole genes (HLA-DRB1/DQB1/DPB1; excluding exon 1 and part of intron 1) by using the AllType NGS kit (One Lambda), Illumina MiSeq platform, and Type Steam Visual NGS analysis software (One Lambda). Results Our validation process comprised 34 NGS runs and 237 samples. In total, 42 samples were analyzed with an overall accuracy and precision of 100% and 100% reproducibility. The overall ambiguity rate was 0% in HLA-A/B/C compared with 56.9% for SBT. Two discrepant alleles of HLA-A and DRB1 were noted in NGS and Sanger sequencing. In accordance with additional SBT exon analyses, one of the results was corrected according to the NGS results. Conclusion NGS HLA typing is a high-throughput approach with high accuracy and precision and a low ambiguity and is promising for use in clinical HLA laboratories.

Reproducibility of next-generation-sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape

Next-generation-sequencing (NGS) becomes increasingly important for laboratories tasked with the detection of genetically modified organisms (GMOs) in food, feed and seeds. Its implementation into standardized workflows demands reliable intra- and inter-laboratory reproducibility. Here, we analyze the reproducibility of short- and long-read targeted NGS and long-read whole genome sequencing (WGS) data between three independent laboratories. Replicate samples were submitted for sequencing and comparatively analyzed. The targeted-NGS-samples consisted of oil seed rape (OSR) sampled from a commodity shipment spiked with a genome edited (GE) OSR and the WGS-samples consisted of leaf material from the GMOs’ parental line. All laboratories delivered highly reproducible high-quality targeted NGS data with little variation. The detection of GMO-related sequences works well regardless of the facility, while the mapping to the complex genome is superior using long read data. Long read WGS is currently not suitable for routine use in enforcement laboratories, due to a large inter-laboratory variation.

Identification and Molecular Characterization of Shamonda Virus in an Aborted Goat Fetus in South Africa.

Viruses in the Orthobunyavirus genus, Peribunyaviridae family, are associated with encephalitis, birth defects and fatalities in animals, and some are zoonotic. Molecular diagnostic investigations of animals with neurological signs previously identified Shuni virus (SHUV) as the most significant orthobunyavirus in South Africa (SA). To determine if other orthobunyaviruses occur in SA, we screened clinical specimens from animals with neurological signs, abortions, and acute deaths from across SA in 2021 using a small (S) segment Simbu serogroup specific TaqMan real-time reverse transcription polymerase chain reaction (RT-PCR). Positive cases were subjected to Sanger sequencing and phylogenetic analysis to identify specific viruses involved, followed by next-generation sequencing (NGS) and additional PCR assays targeting the medium (M) segment and the large (L) segment. In total, 3/172 (1.7%) animals were PCR positive for Simbu serogroup viruses, including two horses with neurological signs and one aborted goat fetus in 2021. Phylogenetic analyses confirmed that the two horses were infected with SHUV strains with nucleotide pairwise (p-) distances of 98.1% and 97.6% to previously identified strains, while the aborted goat fetus was infected with a virus closely related to Shamonda virus (SHAV) with nucleotide p-distances between 94.7% and 91.8%. Virus isolation was unsuccessful, likely due to low levels of infectious particles. However, phylogenetic analyses of a larger fragment of the S segment obtained through NGS and partial sequences of the M and L segments obtained through RT-PCR and Sanger sequencing confirmed that the virus is likely SHAV with nucleotide p-distances between 96.6% and 97.8%. This is the first detection of SHAV in an aborted animal in SA and suggests that SHAV should be considered in differential diagnosis for abortion in animals in Southern Africa.

Confirmation of Insertion, Deletion, and Deletion-Insertion Variants Detected by Next-Generation Sequencing.

Despite clinically demonstrated accuracy in next generation sequencing (NGS) data, many clinical laboratories continue to confirm variants with Sanger sequencing, which increases cost of testing and turnaround time. Several studies have assessed the accuracy of NGS in detecting single nucleotide variants; however, less has been reported about insertion, deletion, and deletion-insertion variants (indels). We performed a retrospective analysis from 2015-2022 of indel results from a subset of NGS targeted gene panel tests offered through the Mayo Clinic Genomics Laboratories. We compared results from NGS and Sanger sequencing of indels observed in clinical runs and during the intra-assay validation of the tests. Results demonstrated 100% concordance between NGS and Sanger sequencing for over 490 indels (217 unique), ranging in size from 1 to 68 basepairs (bp). The majority of indels were deletions (77%) and 1 to 5 bp in length (90%). Variant frequencies ranged from 11.4% to 67.4% and 85.1% to 100% for heterozygous and homozygous variants, respectively, with a median depth of coverage of 2562×. A subset of indels (7%) were located in complex regions of the genome, and these were accurately detected by NGS. We also demonstrated 100% reproducibility of indel detection (n = 179) during intra-assay validation. Together this data demonstrates that reportable indel variants up to 68 bp can be accurately assessed using NGS, even when they occur in complex regions. Depending on the complexity of the region or variant, Sanger sequence confirmation of indels is usually not necessary if the variants meet appropriate coverage and allele frequency thresholds.