Genome Sequencing Approaches Research Articles

Whole genome sequencing approaches for taxonomic profiling and evaluation of wastewater quality

Tracking metagenomic abundance in wastewater is undoubtedly a powerful tool to detect emerging variants and improve community health. However, there are a few factors that limit environmental water-based genomic monitoring: sampling variability, incomplete coverage, genetic fragmentation, degradation, data analysis and interpretation. The decreasing costs of high-throughput sequencing and high-end supercomputers have increased the use and accuracy of genomic data for microbial detection and monitoring in wastewater samples within any given region. To better understand the microbial dynamics and to determine the target sequencing throughput required to establish taxa that may pose as bio-indicators of an epidemiological outbreak, wastewater samples were collected from distinct locations within the Emirate of Abu Dhabi, United Arab Emirates using appropriate sampling methods. A reference database of ∼27,000 known species was developed and used for further analysis. The results showed that 15 % of data in each sample matched any of ∼27,000 known bacterial, viral, fungal, or protozoan species. Despite the high fraction of unclassified data (85 %), more than 2000 species from >800 genera across >30 phyla were detected in each sample. Both 5 Gb and 10 Gb of sequenced data detected the top ∼2000 species with highest abundance. Doubling the target sequencing throughput (i.e., 10 Gb vs 5 Gb) detected ∼500 additional low-abundance species per sample however it did not affect the overall sample composition or translate into higher per-sample species diversity captured. There was a marginal increase in the number of species detected in each sample beyond 0.20 Gb of classified data. Overall, the results indicate that sequencing to a 3 Gb throughput detects nearly 95 % of all species in the samples.

A novel isothermal whole genome sequencing approach for Monkeypox Virus

Monkeypox virus (MPXV) is the zoonotic agent responsible for mpox, an often-self-limiting pox-like disease. Since May 2022, an outbreak characterized by increased human-to-human transmission was detected outside the endemic regions. Whole genome sequencing (WGS) has been successfully used to keep track of viral evolution during outbreaks or for surveillance of multiple pathogens of public health significance. Current WGS protocols for MPXV are either based on metagenomic sequencing or tiled-PCR amplification. The latter allows multiplexing due to the efficient enrichment of the viral DNA, however, mutations or the presence of different clades can negatively influence genome coverage yield. Here, we present the establishment of a novel isothermal WGS method for MPXV based on Phi29 DNA polymerase-based multiple displacement amplification (MDA) properties making use of only 6 primers. This approach yielded from 88% up to 100% genome coverage using either alkaline denatured extracted DNA or clinical material as starting material, with the highest coverage generated by clinical material. We demonstrate that this novel isothermal WGS protocol is suitable for monitoring viral evolution during MPXV outbreaks and surveillance in any conventional laboratory setting.

Development of targeted whole genome sequencing approaches for Crimean-Congo haemorrhagic fever virus (CCHFV)

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, with a reported case fatality rate of 30 % or higher. The virus contains a tri-segmented, negative-sense RNA genome consisting of the small (S), medium (M) and large (L) segments encoding respectively the nucleoprotein (NP), the glycoproteins precursor (GPC) and the viral RNA-dependent RNA polymerase (RDRP). CCHFV is one of the most genetically diverse arboviruses, with seven distinct lineages named after the region they were first reported in and based on S segment phylogenetic analysis.Due to the high genetic divergence of the virus, a single targeted tiling PCR strategy to enrich for viral nucleic acids prior to sequencing is difficult to develop, and previously we have developed and validated a tiling PCR enrichment method for the Europe 1 genetic lineage.We have developed a targeted, probe hybridisation capture method and validated its performance on clinical as well as cell-cultured material of CCHFV from different genetic lineages, including Europe 1, Europe 2, Africa 2 and Africa 3. The method produced over 95 % reference coverages with at least 10x sequencing depth. While we were only able to recover a single complete genome sequence from the tested Europe 1 clinical samples with the capture hybridisation protocol, the data provides evidence of its applicability to different CCHFV genetic lineages.CCHFV is an important tick-borne human pathogen with wide geographical distribution. Environmental as well as anthropogenic factors are causing increased CCHFV transmission. Development of strategies to recover CCHFV sequences from genetically diverse lineages of the virus is of paramount importance to monitor the presence of the virus in new areas, and in public health responses for CCHFV molecular surveillance to rapidly detect, diagnose and characterise currently circulating strains.

Ethical Approaches in Secondary Findings Report from Exome Sequencing Analysis

In Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS) approaches, incidental findings (IFs) or more precisely secondary findings (SFs) have indicated controversial reports. To address SFs issues, well-known guidelines have been released such as the versions of the American College of Medical Genetics and Genomics (ACMG); however, when, to whom, why, and how these SFs should be reported are the key questions that need to be addressed ethically. The current review aimed to investigate the papers with a focus on the ethical approaches regarding IFs/SFs. In this comprehensive review, we searched the PubMed data-base for related publications, and 58 papers were selected as the narrowest reports. There was a positive tendency to disclose the SFs by the professionals and an enthusiasm in patients to be informed about all of their genetic reports. Several studies have addressed that guidelines could not cover all aspects of IFs/SFs ethically and medically. The main focus of these studies was the modernized opt-in informed consent according to some items including prenatal/post-natal or pediatric/adulthood issues. Other foci are genetic variant clinical actionability, cancer status, population-based backgrounds, time- and cost-effectiveness, the education level of sub-jects and clinicians, and the combinations of pharmacogenomics findings to introduce medicinal treatments and personalized prescrip-tions leading to more suitable healthcare. Altogether, concerns remain about the ethical approaches in the SFs; however, the present review introduced remarkable solutions which will be ethically and practically possible to be utilized. This review also found that recent reports are concentrated on the high-volume Exome Sequencing tests of subjects from biobanks and healthcare centers.

How many species are there? Lineage diversification and hidden speciation in Solanaceae from highland grasslands in southern South America.

Species delimitation can be challenging when analysing recently diverged species, especially those taxonomically synonymised due to morphological similarities. We aimed to untangle the relationships between two grassland species, Petunia guarapuavensis and Petunia scheideana, exploring the dynamics of fast divergence and addressing their species delimitation. We used a low-coverage genome sequencing and population genomic approach to distinguish species and populations between P. guarapuavensis and P. scheideana. Our analysis focused on detecting structuration, hybridisation/introgression, and phylogenetic patterns. We employed demographic models to support species delimitation while exploring potential phylogeographic barriers influencing gene flow. Our findings indicated differentiation between the two species and revealed another lineage, which was phylogenetically distinct from the others and had no evidence of gene flow with them. The presence of a river acted as a phylogeographic barrier, limiting gene flow and allowing for structuration between closely related lineages. The optimal species delimitation scenario involved secondary contact between well-established lineages. The rapid divergence observed in these Petunia species explains the lack of significant morphological differences, as floral diagnostic traits in species sharing the pollinators tend to evolve more slowly. This study highlights the complexity of species delimitation in recently diverged groups and emphasises the importance of genomic approaches in understanding evolutionary relationships and speciation dynamics.

Experience of the first adult-focussed undiagnosed disease program in Australia (AHA-UDP): solving rare and puzzling genetic disorders is ageless

BackgroundSignificant recent efforts have facilitated increased access to clinical genetics assessment and genomic sequencing for children with rare diseases in many centres, but there remains a service gap for adults. The Austin Health Adult Undiagnosed Disease Program (AHA-UDP) was designed to complement existing UDP programs that focus on paediatric rare diseases and address an area of unmet diagnostic need for adults with undiagnosed rare conditions in Victoria, Australia. It was conducted at a large Victorian hospital to demonstrate the benefits of bringing genomic techniques currently used predominantly in a research setting into hospital clinical practice, and identify the benefits of enrolling adults with undiagnosed rare diseases into a UDP program. The main objectives were to identify the causal mutation for a variety of diseases of individuals and families enrolled, and to discover novel disease genes.MethodsUnsolved patients in whom standard genomic diagnostic techniques such as targeted gene panel, exome-wide next generation sequencing, and/or chromosomal microarray, had already been performed were recruited. Genome sequencing and enhanced genomic analysis from the research setting were applied to aid novel gene discovery.ResultsIn total, 16/50 (32%) families/cases were solved. One or more candidate variants of uncertain significance were detected in 18/50 (36%) families. No candidate variants were identified in 16/50 (32%) families. Two novel disease genes (TOP3B, PRKACB) and two novel genotype–phenotype correlations (NARS, and KMT2C genes) were identified. Three out of eight patients with suspected mosaic tuberous sclerosis complex had their diagnosis confirmed which provided reproductive options for two patients. The utility of confirming diagnoses for patients with mosaic conditions (using high read depth sequencing and ddPCR) was not specifically envisaged at the onset of the project, but the flexibility to offer recruitment and analyses on an as-needed basis proved to be a strength of the AHA-UDP.ConclusionAHA-UDP demonstrates the utility of a UDP approach applying genome sequencing approaches in diagnosing adults with rare diseases who have had uninformative conventional genetic analysis, informing clinical management, recurrence risk, and recommendations for relatives.

Mitochondrial genetics through the lens of single-cell multi-omics.

Mitochondria carry their own genetic information encoding for a subset of protein-coding genes and translational machinery essential for cellular respiration and metabolism. Despite its small size, the mitochondrial genome, its natural genetic variation and molecular phenotypes have been challenging to study using bulk sequencing approaches, due to its variation in cellular copy number, non-Mendelian modes of inheritance and propensity for mutations. Here we highlight emerging strategies designed to capture mitochondrial genetic variation across individual cells for lineage tracing and studying mitochondrial genetics in primary human cells and clinical specimens. We review recent advances surrounding single-cell mitochondrial genome sequencing and its integration with functional genomic readouts, including leveraging somatic mitochondrial DNA mutations as clonal markers that can resolve cellular population dynamics in complex human tissues. Finally, we discuss how single-cell whole mitochondrial genome sequencing approaches can be utilized to investigate mitochondrial genetics and its contribution to cellular heterogeneity and disease.

Novel liquid biopsy assay to distinguish benign from malignant pelvic masses.

e17563 Background: Despite not ranking among the top ten most common cancers in women in the United States, ovarian cancer stands as the fifth leading cause of cancer-related deaths in this population. According to the American Cancer Society, only 20% of ovarian cancers are found at stages I-II, when 94% of patients are expected to live longer than five years after diagnosis. Instead, the majority of ovarian cancers are detected at stages III-IV, when long term survival is 20% or less. Early identification of ovarian cancer is challenging due to the wide range of symptoms with which a patient may present, most of which lack specificity for ovarian cancer. Even in cases where a physician may want to rule in or out ovarian cancer, the available methods for screening have a low positive predictive value. These methods include bimanual exams, transvaginal ultrasounds, and CA-125 blood tests. In a screening capacity, the sensitivity for a bimanual exam finding ovarian cancer is only about 44%, transvaginal ultrasound is approximately 85%, and CA-125 testing registers at 77%. The need for better tools to detect ovarian cancer is clear. Genece Health has developed an assay utilizing a proprietary algorithm that leverages deep learning to analyze cfDNA fragment size, end motif, and coverage patterns, along with other genomic features, to detect the presence of ctDNA in blood originating from ovarian cancer. The data presented herein demonstrate that the Genece Health assay has the ability to detect ctDNA in a single tube of blood using low pass whole genome sequencing in a cost-effective way to distinguish between malignant and benign pelvic masses. Methods: Blood was prospectively collected from 56 females with pelvic masses in advance of removal and histopathology (approximately 32 cancer and 24 benign masses) in Streck cfDNA BCT Devices. Additionally, healthy female donor blood from 50 individuals procured through the San Diego Blood Bank were collected in Streck cfDNA BCT Devices. cfDNA was extracted from double-spun plasma and low-pass whole genome sequencing (2X – 5X coverage) was performed. Data were analyzed with the Genece Health Algorithm. Results: The Genece Health Algorithm demonstrated the ability to distinguish between pelvic masses that were deemed benign versus malignant via histopathology. There was a significant (P<0.01) difference between the classifier assigned to the malignant and benign and presumed normal groups. Conclusions: The investigational assay described herein shows progress toward the ability to use non-invasive molecular testing to identify ovarian cancer in a blood sample. A blood test to differentiate between a benign and malignant pelvic mass will enhance the ability of physicians to detect ovarian cancer at earlier stage, improving clinical outcomes for women. The cost-effective low-pass whole genome sequencing approach taken by Genece will be further explored in expanded cohorts.

Landscape of genomic alterations in patients with chemoradiation resistant cervical cancer using whole-genome sequencing.

e17531 Background: Cervical cancer is a pressing global health concern and ranks as the second most prevalent female malignancy in India. Unfortunately, 80% of cases are diagnosed at locally advanced stages, leading to significant morbidity and mortality. Standard treatment for locally advanced cervical cancer involves concurrent pelvic radiotherapy and chemotherapy (CCRT). However, a substantial 30%-40% of patients experience CCRT treatment failure. The genomic alterations owing to CCRT resistance are largely unknown. This pilot study aims to identify genomic alterations associated with differential responses to concurrent chemoradiotherapy in cervical cancer patients. Methods: We collected 36 fresh tissue biopsies along with 5 ml of blood from stage IIIB cervical cancer patients. From the total of 36 cervical cancer patients, based on RECIST (Response evaluation criteria in solid tumors) criteria 19 patients were classified as responders and 17 patients were categorized as non-responders to CCRT. Genomic DNA was extracted from both tumor tissue and paired blood samples. Subsequently, whole genome sequencing was performed using the Novaseq 6000 platform, achieving a sequencing depth of 30x-50x coverage with 2x150bp paired reads. The data was further analyzed for Single Nucleotide Variants (SNVs), Copy Number Alterations (CNAs), Structural Variants (SVs), and mutational signatures. Results: In our study, we identified several notable mutated genes, with MUC12 (35%), MUC4 (31%), MACF1 (27%), and OBSCN (27%) being the most prominent. The mutational spectrum encompassed well-known significantly mutated genes such as FBXW7, SPEN, PIK3CA, KMT2C, KMT2D, ARID1A, and STK11, as well as potential novel candidates, genes not previously associated with cervical cancer, such as PKHD1L1, PLEC, and LRP1B. Pathway analysis revealed that several signaling pathways were enriched in cervical cancer, including Notch signaling, Hippo signaling, and RTK/RAS signaling pathways. Mutational signature analysis revealed a prevalence of APOBEC mutagenesis pattern. Copy number alterations displayed amplifications in regions such as 3q, 9p, 14q, and 11p which encompass genes like PIK3CA, JAK2, CD274, and AKT1. Comparative analysis of genomic alterations between chemoradiation resistant and sensitive patients identified mutations in genes related to Notch signaling and TNF signaling, which were exclusively present in the non-responder group. Conclusions: This study represents an initial attempt to understand the genomic landscape of cervical cancer in Indian population using whole genome sequencing approach and its association with responses to CCRT. These findings not only pave the way for further research but also hold promise for the development of targeted therapies, offering potential enhancements in the management of chemoradiation-resistant cervical cancer patients.

Delineating the evolutionary pathway to multidrug-resistant outbreaks of a Mycobacterium tuberculosis L4.1.2.1/Haarlem sublineage

ObjectivesWe sought to capture the evolutionary itinerary of the Mycobacterium tuberculosis L4.1.2.1/Haarlem sublineage in northern Tunisia, where it caused a major multidrug-resistant (MDR) tuberculosis outbreak in a context strictly negative for HIV infection. MethodsWe combined whole genome sequencing and Bayesian approaches using a representative collection of drug-susceptible and drug-resistant L4.1.2.1/Haarlem clinical strains (n = 121) recovered from the outbreak region over 16 years. ResultsIn the absence of drug resistance, the L4.1.2.1/Haarlem sublineage showed a propensity for rapid transmission as witnessed by the high clustering (44.6%) and recent transmission rates (25%), as well as the reduced mean distance between genome pairs. The entire pool of L4.1.2.1/Haarlem MDR strains was found to be linked to either the aforementioned major outbreak (68 individuals, 2001-2016) or to a minor, newly uncovered outbreak (six cases, 2001-2011). Strikingly, the two outbreaks descended independently from a common ancestor that can be dated back to 1886. ConclusionsOur data point to the intrinsic propensity for rapid transmission of the M. tuberculosis L4.1.2.1/Haarlem sublineage in northern Tunisia, linking the overall MDR tuberculosis epidemic to a single ancestor. These findings bring out the important role of the bacillus’ genetic background in the emergence of successful MDR M. tuberculosis clones.

Whole genome sequencing reveals complex resistome features of Klebsiella pneumoniae isolated from patients at major hospitals in Trinidad, West Indies

ObjectivesAntibiotic-resistant Klebsiella pneumoniae is a human pathogen of major global concern due to its ability to cause multiple severe diseases that are often difficult to treat therapeutically. This study aimed to investigate the resistome of local clinical K. pneumoniae isolates. MethodsHerein, we used a whole genome sequencing approach and bioinformatics tools to reconstruct the resistome of 10 clinical K. pneumoniae isolates and one clinical isolate of the closely related Klebsiella quasipneumoniae obtained from patients from three major hospitals in Trinidad, West Indies. ResultsThe results of the study revealed the presence of a complex antibiotic-resistant armoury among the local isolates with multiple resistance mechanisms involving (i) inactivation of antibiotics, (ii) efflux pumps, (iii) antibiotic target alteration, protection, and replacement against antibiotics, and (iv) altered porin protein that reduced the permeability to antibiotics. Several resistance genes such as blaCTX-M-15, blaTEM-1B, blaSHV-28, blaKPC-2, oqxA, sul1, tetD, aac(6′)-Ib-cr5, aph(6)-Id, and fosA6, which are known to confer resistance to antibiotics used to treat K. pneumoniae infections. In most cases, the resistance genes were flanked by mobile elements, including insertion sequences and transposons, which facilitate the spread of these genetic features among related organisms. ConclusionThis is the first comprehensive study to thoroughly investigate the resistome of clinical K. pneumoniae isolates and K. quasipneumoniae from Trinidad, West Indies. These findings suggest that monitoring K. pneumoniae and its genome-wide antibiotic resistance features in clinical strains would be of critical importance for guiding antibiotic stewardship programs and improving regional disease management systems for this pathogen.

Exploring the interactions between algae and archaea

AbstractAlgae and archaea co-exist in diverse aquatic ecosystems and play a significant role in ecological functions and biogeochemical cycles. Compared to well-studied algal–bacterial interactions, there is a lack of information on algal–archaeal interactions and how their interactions affect their physiological fitness and nutrient cycles in either artificial cultivation systems or natural environments. The vast archaeal biodiversity, as indicated by genomic sequencing and computational approaches, has stimulated great interest in exploring uncultivated archaea to expand our knowledge of algae-archaea symbiosis. In this review, we summarize the latest studies on the diversity of algae-associated archaea and their (putative) symbiotic interactions, highlight the effects of algal–archaeal interactions on biogeochemical cycles and extend such knowledge to facilitate novel archaeal isolation and a broad range of algae-based biotechnological applications.

Abstract CT257: Hyperprogressive disease following bintrafusp alfa and DNA damaging chemotherapy in relapsed small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive type of lung cancer. Current therapies have limited efficacy in SCLC, and despite a highly mutated genome, SCLC is largely unresponsive to immunotherapy. We sought to leverage the immune surveillance triggered by DNA damage in patients with relapsed SCLC using bintrafusp alfa, a bifunctional fusion protein targeting both PD-L1 and TGF-beta. Hyperprogressive disease (HPD), characterized by the rapid acceleration of tumor growth, has been reported in approximately 13% of patients treated with immune checkpoint inhibitors. However, the mechanistic basis is poorly understood. Methods: This is a safety run-in and phase II clinical trial. Cohort 1 enrolled patients with relapsed SCLC across Arms A (bintrafusp alfa 2400 mg q3 wks), B (bintrafusp alfa 2400 mg on D1 plus topotecan 1 mg/m2/day on D1-5 q3 wks), and C (bintrafusp alfa 1200 mg on D1 q2 wks plus temozolomide 200 mg/m2/day on D1-5 q4 wks). The primary endpoint was objective response rate (ORR). Results: Thirty-seven patients enrolled. Grade 3 treatment-related adverse events (TRAE) included transaminitis (27.0%), anemia (24.4%), lymphopenia (10.8%), and maculopapular rash (5.4%). Grade 4 TRAE included thrombocytopenia (8.1%), lymphopenia (5.4%) and transaminitis (5.4%). One patient experienced grade 5 tumor hemorrhage. By RECIST 1.1, partial responses were observed in 5 (13.5%), stable disease in 11 (29.7%), and progressive disease in 21 (56.8%) patients. Importantly, 13 (35.1%) patients developed HPD (≥ 2x increase in tumor growth rate from pre-trial vs on-trial, time to treatment failure ≤ 2 months, and ≥ 50% increase tumor burden). Changes in circulating cell-free DNA (n=20) tumor fraction using DELFI-TF, a mutation-independent, low-coverage whole genome sequencing approach, correlated with treatment responses. Pre-treatment serum from HPD (n=10 vs. 9 non-HPD) patients showed significantly lower levels of inflammatory cytokines (CXCL10, CCL13, CCL8, CCL19, TRAIL) and Granzyme H, and higher levels of anti-inflammatory cytokine IL10. Consistently, pre-treatment tumor transcriptomes of HPD (n=5 vs. 7 non-HPD) patients revealed suppression of IFN-gamma response, allograft rejection, and inflammatory response pathways. Conclusions: Concomitant TGF-beta and PD-L1 blockade is associated with a high frequency of HPD in SCLC patients. cfDNA could be critical for monitoring HPD. Tumor and blood immune signatures may inform the likelihood of HPD, with immune excluded tumors more likely to develop HPD. NCT Number: NCT03554473 Bintrafusp alfa was provided by EMD Serono (CrossRef Funder ID: 10.13039/100004755) Citation Format: Brett Schroeder, Nobuyuki Takahashi, Howard Yang, Renee Donahue, Zachary Skidmore, Alissa Konicki, Michael Nirula, Max Greenberg, George Chrisafis, Yang Zhang, Yo-Ting Tsai, Linda Sciuto, Samantha Nichols, Melissa Abel, Parth Desai, Rajesh Kumar, Christopher Schultz, Danielle Pinkiert, Chante Graham, Ajit Sharma, Justin Malin, Manan Krishnamurthy, Sophie Zhuang, Maxwell Lee, Lorenzo Rinaldi, Jeffrey Schlom, Lalage Wakefield, Anish Thomas. Hyperprogressive disease following bintrafusp alfa and DNA damaging chemotherapy in relapsed small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT257.

Abstract 3898: SJPedPanel: A pan-cancer gene panel for childhood malignancies

Abstract Background: Extensive efforts in the past decade have revolutionized our understanding of the genetic underpinnings of childhood malignancies and identified numerous driver alterations that can provide potential targets for novel therapy and are excellent biomarkers for disease monitoring. For these purposes, a whole genome or exome sequencing approach can be resource prohibitive. Numerous gene panels are developed for adult cancers to address these challenges. Due to the dramatic differences in driver gene landscapes between pediatric and adult cancers, a gene panel for childhood cancers is needed. Results: Here, we have developed a gene panel dedicated to childhood cancers. This panel (2.8 Mbp) covers 5275 coding exons of 357 driver genes, 297 introns frequently involved in rearrangements that generate fusion oncoproteins, commonly deleted regions, such as CDKN2A and PAX5 (for B-/T-ALL) and SMARCB1 (for ATRT), and 7,590 polymorphism sites to detect copy number alterations for interrogating tumors with aneuploidy, such as hyperdiploid and hypodiploid B-ALL or 17q gain neuroblastoma. We used driver alterations reported from an established real-time clinical genomics cohort (n=253) to investigate the effectiveness of this gene panel. Among the 485 pathogenic variants reported, our panel covered 417 variants (86%). For 90 rearrangements responsible for oncogenic fusions, our panel covered 74 events (82%). We re-sequenced 113 previously characterized clinical specimens at an average depth of 2,500X using SJPedPanel and recovered 355 (90%) of the 396 reported pathogenic variants. Among the 30 unique genes of the 41 missed alterations, 29 genes are mutated in pediatric cancers with a low frequency (<0.21%) and hence not covered in the panel. We then investigated the power of this panel in detecting mutations from specimens with low tumor content (as low as 0.1%) using cell line-based dilution experiments and discovered that this gene panel enabled us to detect ~80% variants with allele fraction of 0.2%, while the detection rate decreases to ~50% when the allele fraction is 0.1%. We finally demonstrate its utility in disease monitoring on clinical specimens collected from AML patients in morphologic remission. Conclusion: Overall, our gene panel enables the detection of clinically relevant genetic alterations including rearrangements responsible for subtype-defining fusions for childhood cancers by targeted sequencing of ~0.15% of human genome. Our panel will significantly enhance the analysis of specimens with low tumor burdens for cancer monitoring and early detection. Citation Format: Pandurang Kolekar, Vidya Balagopal, Li Dong, Yanling Liu, Scott Foy, Quang Tran, Heather Mulder, Anna LW Huskey, Emily Plyler, Zhikai Liang, Jingqun Ma, Joy Nakitandwe, Jiali Gu, Jamie Maciaszek, Debbie Payne-Turner, Saradhi Mallampati, Lu Wang, John Easton, Jeffery M. Klco, Xiaotu Ma. SJPedPanel: A pan-cancer gene panel for childhood malignancies [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 3898.

Implementation of a high-throughput whole genome sequencing approach with the goal of maximizing efficiency and cost effectiveness to improve public health.

Public Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Shiga toxin-producing Escherichia coli isolated from hunted wild boar (Sus scrofa) in Switzerland.

Shiga toxin-producing Escherichia (E.) coli (STEC) are zoonotic foodborne pathogens of significant public health importance. While ruminants are considered the main reservoir, wild animals are increasingly acknowledged as carriers and potential reservoirs of STEC. The aim of this study was to determine the occurrence of STEC in a total of 59 faecal samples of hunted wild boars (Sus scrofa) from two different regions in Switzerland (canton Thurgau in northern Switzerland and canton Ticino in southern Switzerland), and to characterise the isolates using a whole genome sequencing approach. After an enrichment step, Shiga-toxin encoding genes (stx) were detected by real-time PCR in 41 % (95 % confidence interval (95 %CI) 0,29 - 0,53) of the samples, and STEC were subsequently recovered from 22 % (95 %CI 0,13 - 0,34) of the same samples. Seven different serotypes and six different sequence types (STs) were found, with O146:H28 ST738 (n = 4) and O100:H20 ST2514 (n = 4) predominating. Subtyping of stx identified isolates with stx1c/stx2b (n = 1), stx2a (n = 1), stx2b (n = 6), and stx2e (n = 6). No isolate contained the eae gene, but all harboured additional virulence genes, most commonly astA (n = 10), hlyE (n = 9), and hra (n = 9). STEC O11:H5, O21:H21, and O146:H28 harboured virulence factors associated with extra-intestinal pathogenic E. coli (ExPEC), and STEC O100:H20 and O155:H26 possessed sta1 and/or stb and were STEC/enterotoxigenic E. coli (ETEC) hybrid pathotypes. Our results show that wild boars are carriers of STEC which may be distributed in the environment, possibly leading to the contamination of agricultural crops and water sources. The serogroups included STEC O146 which belongs to the most common non-O157 serogroups associated with human illness in Europe, with implications for public health. Since Stx2e-producing STEC have frequently been reported in swine and pork, STEC O100:H20 harbouring stx2e in faeces of wild boars may be relevant to free-range systems of pig farming because of the potential risk of transmission events at thewildlife-livestock interface.

A Mini-review on Helicobacter pylori with Gastric Cancer and Available Treatments.

Helicobacter pylori (H. pylori) is the most thoroughly researched etiological component for stomach inflammation and malignancies. Even though there are conventional recommendations and treatment regimens for eradicating H. pylori, failure rates continue to climb. Antibiotic resistance contributes significantly to misdiagnoses, false positive results, and clinical failures, all of which raise the chance of infection recurrence. This review aims to explore the molecular mechanisms underlying drug resistance in H. pylori and discuss novel approaches for detecting genotypic resistance. Modulation of drug uptake/ efflux, biofilm, and coccoid development. Newer genome sequencing approaches capable of detecting H. pylori genotypic resistance are presented. Prolonged infection in the stomach causes major problems such as gastric cancer. The review discusses how H. pylori causes stomach cancer, recent biomarkers such as miRNAs, molecular pathways in the development of gastric cancer, and diagnostic methods and clinical trials for the disease. Efforts have been made to summarize the recent advancements made toward early diagnosis and novel therapeutic approaches for H. pylori-induced gastric cancer.

Whole genome sequence-based characterization of Campylobacter isolated from broiler carcasses over a three-year period in a big poultry slaughterhouse reveals high genetic diversity and a recurring genomic lineage of Campylobacter jejuni

Campylobacter is among the most frequent agents of bacterial gastroenteritis in Europe and is primarily linked to the consumption of contaminated food. The aim of this study was to assess genomic diversity and to identify antimicrobial resistance and virulence genes of 155 Campylobacter isolated from broiler carcasses (neck skin samples) in a large-scale Swiss poultry abattoir over a three-year period. Samples originated from broilers from three different types of farming systems (particularly animal-friendly stabling (PAFS), free-range farms, and organic farms).Campylobacter jejuni (n = 127) and Campylobacter coli (n = 28) were analysed using a whole genome sequencing (WGS) approach (MiniSeq; Illumina). Sequence types (STs) were determined in silico from the WGS data and isolates were assigned into complex types (CTs) using the cgMLST SeqSphere+ scheme. Antimicrobial resistance genes were identified using the Resistance Gene Identifier (RGI), and virulence genes were identified using the virulence factor database (VFDB).A high degree of genetic diversity was observed. Many sequence types (C. jejuni ST19, ST21, ST48, ST50, ST122, ST262 and C. coli ST827) occurred more than once and were distributed throughout the study period, irrespective of the year of isolation and of the broiler farming type. Antimicrobial resistance determinants included blaOXA and tet(O) genes, as well as the T86I substitution within GyrA. Virulence genes known to play a role in human Campylobacter infection were identified such as the wlaN, cstIII, neuA1, neuB1, and neuC1. Subtyping of the Campylobacter isolates identified the occurrence of a highly clonal population of C. jejuni ST21 that was isolated throughout the three-year study period from carcasses from farms with geographically different locations and different farming systems.The high rate of genetic diversity observed among broiler carcass isolates is consistent with previous studies. The identification of a persisting highly clonal C. jejuni ST21 subtype suggests that the slaughterhouse may represent an environment in which C. jejuni ST21 may survive, however, the ecological reservoir potentially maintaining this clone remains unknown.

Unravelling the genomic origins of lumpy skin disease virus in recent outbreaks.

Lumpy skin disease virus (LSDV) belongs to the genus Capripoxvirus and family Poxviridae. LSDV was endemic in most of Africa, the Middle East and Turkey, but since 2015, several outbreaks have been reported in other countries. In this study, we used whole genome sequencing approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations compared to the Reference Neethling strain sequence and the previous field strains. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. Thirty-eight genes have more than 2 variations per gene, and these genes belong to viral-core proteins, viral binding proteins, replication, and RNA polymerase proteins. We highlight the importance of several SNPs in various genes, which may play an essential role in the pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

Metabolic analysis of the CAZy class glycosyltransferases in rhizospheric soil fungiome of the plant species Moringa oleifera

The target of the present work is to study the most abundant carbohydrate-active enzymes (CAZymes) of glycosyltransferase (GT) class, which are encoded by fungiome genes present in the rhizospheric soil of the plant species Moringa oleifera. The datasets of this CAZy class were recovered using metagenomic whole shotgun genome sequencing approach, and the resultant CAZymes were searched against the KEGG pathway database to identify function. High emphasis was given to the two GT families, GT4 and GT2, which were the highest within GT class in the number and abundance of gene queries in this soil compartment. These two GT families harbor CAZymes playing crucial roles in cell membrane and cell wall processes. These CAZymes are responsible for synthesizing essential structural components such as cellulose and chitin, which contribute to the integrity of cell walls in plants and fungi. The CAZyme beta-1,3-glucan synthase of GT2 family accumulates 1,3-β-glucan, which provides elasticity as well as tensile strength to the fungal cell wall. Other GT CAZymes contribute to the biosynthesis of several compounds crucial for cell membrane and wall integrity, including lipopolysaccharide, e.g., lipopolysaccharide N-acetylglucosaminyltransferase, cell wall teichoic acid, e.g., alpha-glucosyltransferase, and cellulose, e.g., cellulose synthase. These compounds also play pivotal roles in ion homeostasis, organic carbon mineralization, and osmoprotection against abiotic stresses in plants. This study emphasizes the major roles of these two CAZy GT families in connecting the structure and function of cell membranes and cell walls of fungal and plant cells. The study also sheds light on the potential occurrence of tripartite symbiotic relationships involving the plant, rhizospheric bacteriome, and fungiome via the action of CAZymes of GT4 and GT2 families. These findings provide valuable insights towards the generation of innovative agricultural practices to enhance the performance of crop plants in the future.