Whole Genome Sequencing Research Articles

Tracking SARS-CoV-2 RNA in the Air: Lessons from a COVID-19 Outbreak in an Infirmary Unit

BackgroundThe duration and magnitude of SARS-CoV-2 air dispersal during nosocomial outbreaks remain uncertain. This study evaluates the impact of mobile modular high-efficiency-particulate-air filter units (MMHUs) on SARS-CoV-2 air dispersal. MethodsWe investigated a nosocomial COVID-19 outbreak in an infirmary unit. The viral load (VL) of SARS-CoV-2 in air samples was correlated with the VL in nasopharyngeal swabs (NPS) with or without MMHU. These samples underwent whole genome sequencing (WGS) and phylogenetic analysis. ResultsUpon outbreak declaration (2 August 2024, Day 0), 44 (69.8%) of 63 patients acquired COVID-19 in Ward 2B (19 male) and 2C (25 female) by Day 4. The VL of SARS-CoV-2 remained detectable in air until Day 11 (2B) and Day 20 (2C). The VL of air samples was significantly correlated with the VL in NPS collected on Days 5,7,10, and 13 in 2C (r= -0.975, p=0.004). Using MMHU, the mean daily ratio of SARS-CoV-2 RNA (copies/1,000L of air/patient) in 2B was five times lower than in 2C from Days 5 to 10. WGS revealed all 41 tested strains belonged to the Omicron variant, KP.3.1.1, phylogenetically related to the prevailing community strains. ConclusionUsing MMHU mitigates the duration and magnitude of SARS-CoV-2 air dispersal during nosocomial outbreak.

First Complete Genome Sequence of Nigrospora osmanthi: A Fundamental Resource for Studying the Plant Pathogenic Fungus

Nigrospora osmanthi strain Z1 is a potential bioherbicide for the biological control of water lettuce, one of the most common invasive weeds. Here, the first high-quality genome assembly of N. osmanthi was obtained by a combination of PacBio and Illumina sequencing technologies. Twelve contigs representing the nuclear genome and one mitochondrial DNA were generated, with a total length of 45.94 Mb, an N50 equal to 6.99 Mb, and a GC content of 57.91%; 9 of the 12 nuclear contigs represented chromosomes from telomere to telomere characterized by telomeric-like repeats. A total of 11,051 genes were predicted using a combination of gene prediction tools, including 336 noncoding RNAs, 1,298 genes encoding secretory proteins, 1,735 genes encoding carbohydrate-active enzymes, 1,955 fungal virulence factors, and 50 putative secondary metabolite gene clusters. This complete genome sequence can serve as a valuable resource for future comparative genomic studies of the fungus Nigrospora for effective application in the biocontrol of invasive species and the discovery of fungal secondary metabolites. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Genome-wide identification and expression analysis of SpUGE gene family and heterologous expression-mediated Arabidopsis thaliana tolerance to Cd stress

The UDP-glucose 4-epimerase (UGE) enzyme plays a critical role in plant growth and responses to abiotic stressors, such as heavy metal exposure. However, UGE-mediated remodeling of cell wall polysaccharides in response to these stressors remains poorly understood in willow. This study investigated the structure, function, and expression patterns of the UGE gene family in willow, focusing on cadmium treatment to elucidate how SpUGE1 enhances Cd resistance. Six SpUGE genes were identified through whole-genome sequencing and bioinformatics analysis, and they were mapped across five chromosomes. Quantitative PCR analysis revealed that, with the exception of SpUGE3, all genes showed their highest relative expression in the leaves. Under Cd treatment, members of the SpUGE gene family displayed varying levels of responsiveness, with SpUGE1 showing a marked increase in expression over time. In transgenic Arabidopsis thaliana overexpressing SpUGE1, the cellulose, hemicellulose, lignin, and pectin content significantly increased, with cellulose levels rising by >50 % and pectin by approximately 30 %. This overexpression conferred enhanced Cd resistance by increasing cell wall thickness through elevated cell wall polysaccharides, which reduced Cd uptake. Consequently, Cd content in the cell wall, chloroplasts, and mitochondria was significantly lower than that in wild-type plants, reducing cellular damage and improving Cd resistance. Overall, this study provides valuable theoretical and experimental insights into the role of the SpUGE1 gene family in willow.

Serratia marcescens outbreak in a neonatal intensive care unit associated with contaminated handwashing sinks

PurposeThis article describes the origin of a S. marcescens outbreak in a neonatal intensive care unit (NICU). Materials and methodsA retrospective case-control study including 12 S. marcescens-positive and 22 S. marcescens-negative neonates in the NICU was performed to identify the source of the outbreak. S. marcescens isolates were collected during the outbreak and analyzed using whole-genome sequencing (WGS). IQ-Tree software, BEAST2 software package and SCOTTI software were used to construct a phylogenetic tree and a propagation path map. ResultsThe index case occurred on February 21st and outbreak ended on March 9th, 2021, affecting a total of 12 neonates (2 with S. marcescens infection and 10 with S. marcescens colonization). Multivariate logistic regression identified that the distance of <0.8 m between the bed unit and the sink (odds ratio [OR], 20.50; 95 % confidence interval [CI], 1.09–384.86), a large number of rotating nurses within a week (OR 2.58, 95 % CI, 1.09–6.11) and use of humidification water in the incubator (OR 189.70, 95 % CI, 2.76–13027.31) were significant increased risk factors for S. marcescens infection or colonization in the outbreak. WGS sifted out a predominant clone between contaminated handwashing sinks and patients, suggesting that cross-transmission was involved in the dissemination of S. marcescens. ConclusionContaminated handwashing sinks can be a communication intermediary of S. marcescens infection or colonization of neonates in the NICU. A distance of <0.8 m between the bed unit and the sink, and a large number of rotating nurses might play important roles in this outbreak. Attention should be paid to sinks contamination and contact transmission to prevent outbreaks.

Microbiological Aspects and Clinical Impact of Lower Lung Field Tuberculosis: an Observational Cohort Study in Peru.

Approximately 40% of tuberculosis (TB) cases remain undiagnosed globally. Lower lung field tuberculosis (LLF TB) presents atypically and is often misidentified as other lung diseases, leading to diagnostic delays in resource-limited settings. It may contribute to increased TB transmission and mortality. We aimed to identify microbiological determinants of LLF TB and evaluate treatment responses to optimize care. We conducted an observational cohort study in Lima, Peru, enrolling adults with microbiologically confirmed pulmonary TB (PTB) diagnosed by GeneXpert MTB/RIF assay or sputum culture. Mycobacterium tuberculosis (MTB) lineage was determined using whole-genome sequencing (WGS). Due to the delayed chest radiograph (CXR) changes in LLF TB compared to non-LLF TB, we measured changes in the St. George's Respiratory Questionnaire (SGRQ) score at two-month treatment mark and evaluated World Health Organization (WHO)-specified final treatment outcomes. We used logistic regression to evaluate the associations between LLF TB and microbiological determinants and treatment outcomes. We used linear regression to assess whether the change in SGRQ scores over the first 2 months of treatment varied by LLF TB status. Among 1,316 PTB patients, 84 (6%) had LLF TB. Compared to non-LLF TB patients, LLF TB patients were more likely to be smear-negative (adjusted odds ratio [aOR] [95% CI]=2.04 [1.28-3.23], p=0.002) and to be infected with Lineage 2 (aOR [95% CI]= 1.95 (95% CI: 1.07 to 3.41; p=0.024). People with LLF TB had less improvement in SGRQ scores after two months of treatment (adjusted score difference [95% CI] = -6.29 [-10.99 to -1.59], p = 0.009), while they experienced better final outcomes compared to non-LLF TB patients, though this difference did not reach statistical significance (adjusted odds ratio [aOR] = 0.43 [95% CI: 0.13 to 1.05], p = 0.103). Patients with LLF TB are more likely than those with upper lung lesions to be sputum-negative on conventional tests, to be infected with Lineage 2, and to show less clinical improvement after treatment.

Screening, identification, engineering, and characterization of Bacillus-derived α-amylase for effective tobacco starch degradation

In this study, two high-performing α-amylase-producing strains, CK3–5 and A8–1 were successfully isolated and characterized, which were taxonomically confirmed as Bacillus velezensis through whole-genome sequencing and bioinformatics. Bioinformatic sequence analysis and molecular docking revealed the catalytic triad (Asp173-Glu208-Asp274) essential for α-amylase function. Through metabolic engineering, the recombinant strain BAX-5/PT17amy(A8–1)SP002 was developed, which exhibited the highest α-amylase activity of 1440 U/mL upon fermentation optimization, marking a 9.2-fold enhancement over the wild-type strain A8–1, and it successfully degraded 6 % of the starch in the tobacco leaves within 48 h, while the content of 13 harmful substances, including acetamide, pyridine, and acetonitrile, was reduced by 8.6 % to 25.2 %. This study reveals a novel α-amylase gene from B. velezensis and establishes an efficient expression system in B. amyloliquefaciens, offering valuable insights for industrial α-amylase production.

Whole-Genome Analysis of Stress Resistance-Related Genes in Listeria monocytogenes

Listeria monocytogenes is a crucial foodborne pathogen with significant public health implications. This study analyzed whole-genome sequences (WGS) of L. monocytogenes strains from public databases, examining associations between resistance genes, lineage, strain type, isolation source, and geography. Results revealed that after eliminating duplicates and strains with incomplete WGS, a total of 316 strains were deemed suitable for subsequent analyses. Within these strains, lineages I and II were extensively distributed, predominantly isolated from clinical and food sources. 56.65% of these strains fell into seven major Clonal Complexes (CC), identified by Multilocus Sequence Typing (MLST), correlating significantly with isolation information. Analysis of 46 resistance-related genes showed a high consistency of resistance genes in the same type of strains, hinting at a potential causal chain of ‘food-food environment-evolution of L. monocytogenes’. Moreover, the standard strains exhibit similar gene carriage rates as the sample strains, with multiple variations observed in acid-resistance genes. In conclusion, through a comprehensive analysis of the L. monocytogenes genome sequences, this study deepens our understanding of the differences and associations between its lineage, strain typing, isolation sources, geographical distribution, and resistance genes, thereby providing a scientific basis for formulating more effective prevention and control strategies.

Genomes and epigenomes of matched normal and tumor breast tissue reveal diverse evolutionary trajectories and tumor-host interactions.

Normal tissues adjacent to the tumor (NATs) may harbor early breast carcinogenesis events driven by field cancerization. Although previous studies have characterized copy-number (CN) and transcriptomic alterations, the evolutionary history of NATs in breast cancer (BC) remains poorly characterized. Utilizing whole-genome sequencing (WGS), methylation profiling, and RNA sequencing (RNA-seq), we analyzed paired germline, NATs, and tumor samples from 43 individuals with BC in Hong Kong (HK). We found that single-nucleotide variants (SNVs) were common in NATs, with one-third of NAT samples exhibiting SNVs in driver genes, many of which were present in paired tumor samples. The most frequently mutated genes in both tumor and NAT samples were PIK3CA, TP53, GATA3, and AKT1. In contrast, large-scale aberrations such as somatic CN alterations (SCNAs) and structural variants (SVs) were rarely detected in NAT samples. We generated phylogenetic trees to investigate the evolutionary history of paired NAT and tumor samples. They could be categorized into tumor only, shared, and multiple-tree groups, the last of which is concordant with non-genetic field cancerization. These groups exhibited distinct genomic and epigenomic characteristics in both NAT and tumor samples.Specifically, NAT samples in the shared-tree group showed higher number of mutations, while NAT samples belonging to the multiple-tree group showed a less inflammatory tumor microenvironment (TME), characterized by a higher proportion ofregulatory Tcells (Tregs) and lower presence of CD14 cell populations. In summary, our findings highlight the diverse evolutionary history in BC NAT/tumor pairs and the impact of field cancerization and TME in shaping the genomic evolutionary historyof tumors.

Binding of PtoRAP2.12 to demethylated and accessible chromatin regions in the PtoGntK promoter stimulates growth of poplar.

DNA methylation is an essential epigenetic modification for gene regulation in plant growth and development. However, the precise mechanisms of DNA methylation remain poorly understood, especially in woody plants. We employed whole-genome bisulfite sequencing (WGBS), assays for transposase-accessible chromatin using sequencing (ATAC-seq), and RNA-Seq to investigate epigenetic regulatory relationships in Populus tomentosa treated with DNA methylation inhibitor 5-azacitidine. Expression-quantitative trait methylation analysis (eQTM), epigenome-wide association study (EWAS), and joint linkage-linkage disequilibrium mapping were used to explore the epigenetic regulatory genes, and using CRISPR/Cas9 to identify the role of candidate genes. Plant developmental abnormalities occurred when DNA methylation levels were substantially reduced. DNA methylation regulated 112 expressed genes via chromatin accessibility, of which 61 genes were significantly influenced by DNA methylation variation at the population level. One DNA methylation-regulated gene, PtoGntK, was located in a major quantitative trait locus (QTL) for poplar growth. Overexpression and CRISPR/Cas9 of PtoGntK revealed it affected poplar height and stem diameter. The PtoRAP2.12 was found to bind to the demethylated accessible region in the PtoGntK promoter, thereby promoting growth in poplar. This study identified key genes with epigenetic regulation for plant growth and provides insights into epigenetic regulation mechanisms in woody plants.

Genomics-based timely detection of Dengue Virus type I genotypes I and V in Uruguay

This study details a genomics-based approach for the early detection of mosquito-borne pathogens, marked by Uruguay's first ever complete genomic sequencing of Dengue Virus type I genotypes I and V. This pioneering effort has facilitated the prompt identification of these genotypes within the country, enabling Uruguayan public health authorities to develop timely and effective response strategies. Further integrated into this approach is a climate-driven suitability measure, closely associated with Dengue case reports and indicative of the local climate's role in the virus's transmission in the country within the changing climate context. The detection of multiple DENV-1 genotypes co-circulating in Uruguay underscores the necessity for proactive surveillance, particularly at borders, to prevent the introduction and dissemination of novel viral strains within the country and the region. This approach aids in facilitating prompt public health responses and intervention strategies, which are crucial in mitigating the impact of dengue outbreaks.

Complete genome sequences of two novel Ralstonia jumbo phages isolated from leaf litter compost

Two Ralstonia phages, FLC1-1B and FLC4-3B, were isolated from leaf litter compost, using Ralstonia pseudosolanacearum, which is a causal agent of bacterial wilt disease, as a host. The genomic DNA sequences of FLC1-1B and FLC4-3B were determined and found to be 290,008 bp and 291,257 bp in length, respectively, and they were therefore classified as jumbo phages. However, they did not show high similarity to any jumbo phage genomic sequence in the NCBI nt database. The closest hit in a BLAST search was the jumbo phage ripduovirus RP12, with only 35% coverage and 77% sequence identity, whereas the FLC1-1B and FLC4-3B sequences were 99.0% identical. Based on these findings, FLC1-1B and FLC4-3B should be classified as members of a new genus in the order Caudoviricetes. FLC4-3B was found to suppress wilt disease in tomato plants, suggesting that it has potential as a biocontrol agent for managing R. pseudosolanacearum infections.

Isolation and characterization of Bifidobacterium spp. From breast milk with different human milk oligosaccharides utilization and anti-inflammatory capacity

Breast milk is the best source of nutrition for infants. Human milk oligosaccharides (HMOs) and the corresponding HMOs-consuming Bifidobacterium positively influence infant health. This study aims to isolate and characterize Bifidobacterium from breast milk of healthy Chinese mothers, identifying the most efficacious strains for inclusion in simulated maternal milk formulas. Nine Bifidobacterium strains (two of B. breve and seven of B. infantis) were isolated, exhibiting a broad spectrum of probiotic potential. This included tolerance to simulated infant gastrointestinal conditions, notable adhesion, antibacterial, antioxidant activities, and HMOs utilization ability. Lacto-N-Tetraose (LNT) is preferred in early growth among Bifidobacterium isolates. B. breve showed a preference for LNT, whereas B. infantis showed a preference for fucosylated HMOs, and displayed reduced utilization of sialylated HMOs. They also exhibited robust safety profiles, including no hemolytic activity, an appropriate D/L lactate-producing ratio, and non-toxicity in an acute oral toxicity assay on mice. It is noteworthy that B. breve N-90, O-147, B. infantis O-161 and R-1 exhibited anti-inflammatory effects in LPS-induced RAW 264.7 cells. Specifically, a notable reduction in TNF-α levels was observed in pre-treatment, while a decrease in IL-1β and IL-6 levels in co-treatment. B. breve N-90 and B. infantis R-1 were identified finally as promising probiotic candidates. Their whole-genome sequencing analysis confirmed presence of functional genes associated with gastrointestinal colonization, antioxidation, and glycoside hydrolase activity on HMOs. The annotation for antibiotic resistance and virulence genes concurred with phenotypes, further validating the safety. Breast milk is a good source for Bifidobacteria isolation, while Bifidobacteria utilize HMOs in a strain-dependent manner. The two selected strains, B. breve N-90 and B. infantis R-1, are potential candidates for inclusion in simulated maternal milk formulas and deserved further in vivo investigation for their health-promoting effects.

Research Note: Whole-genome sequencing revealed genomic diversity dynamics in duck conserved populations

The purpose of this study was to comprehensively analyze the genetic diversity of the Pekin duck conserved population with five generations and to evaluate the effectiveness of the current conservation strategy. In total, 277 Pekin duck conserved individuals and 40 Mallards as ancestral controls were collected. Each duck was sequenced at about 10X whole-genome coverage, while over 7.4 million single nucleotide polymorphisms (SNPs) in total were detected for genetic diversity analysis. Both the expected heterozygosity and observed heterozygosity values exceeded 0.3. The genetic differentiation (FST) values ranged from 0.007 to 0.039, and the Polymorphism information content (PIC) values ranged from 0.29 to 0.34. These results indicate no significant differentiation between generations, and the genetic diversity remains high. In particular, the inbreeding coefficient has been strictly controlled and has not increased rapidly during the conservation. Overall, the inbreeding coefficient of the conserved Pekin duck population was higher than that of its wild ancestors, indicating that domestication has resulted in reduced genetic diversity. This is the first report using whole genome resequencing data to systematically evaluate the genomic dynamics across several generations in ducks. The results show that the strategy of free mating and random seed retention within sire families is effective for maintaining the genetic diversity of the conserved Pekin duck population.

Genomic Epidemiology of Human Respiratory Syncytial Virus, Minnesota, USA, July 2023-February 2024.

We recently expanded the viral genomic surveillance program in Minnesota, USA, to include human respiratory syncytial virus. We performed whole-genome sequencing of 575 specimens collected at Minnesota healthcare facilities during July 2023-February 2024. Subgroups A and B differed in their genomic landscapes, and we identified 23 clusters of genetically identical genomes.

Sequencing and Analysis of Wolbachia Strains from A and B Supergroups Detected in Sylvatic Mosquitoes from Brazil

Wolbachia are endosymbiotic bacteria that infect a wide range of arthropods and filarial nematodes, often manipulating host reproduction. The efficacy of Wolbachia-based interventions for dengue and chikungunya control has been validated through numerous field studies in recent years. This study aimed to investigate the diversity and prevalence of Wolbachia infections in sylvatic mosquitoes from two locations in Recife, Brazil. Multiple mosquito species were screened for Wolbachia using both target marker gene amplification coupled with Sanger sequencing and whole-genome sequencing (WGS) approaches. Phylogenetic analyses were conducted to classify Wolbachia strains into supergroups and assess their evolutionary relationships. Results revealed the presence of Wolbachia in eleven mosquito species examined, with different infection rates. Both supergroups A and B of Wolbachia strains were identified, with Aedes albopictus showing co-infection by both supergroups through the WGS approach. We also detected indirect evidence of Wolbachia horizontal transmission among mosquitoes and other distant host orders. This study provides valuable insights into the distribution and diversity of Wolbachia in sylvatic mosquitoes from Brazil and adds new important data about Wolbachia detection through target marker gene amplicon coupled with Sanger sequencing and WGS methods, highlighting its complementarity to ascertain the presence of Wolbachia in mosquito samples.

An Oxford Nanopore Technology-Based Hepatitis B Virus Sequencing Protocol Suitable for Genomic Surveillance Within Clinical Diagnostic Settings

Chronic Hepatitis B Virus (HBV) infection remains a significant public health concern, particularly in Africa, where the burden is substantial. HBV is an enveloped virus, classified into ten phylogenetically distinct genotypes (A–J). Tests to determine HBV genotypes are based on full-genome sequencing or reverse hybridization. In practice, both approaches have limitations. Whereas diagnostic sequencing, generally using the Sanger approach, tends to focus only on the S-gene and yields little or no information on intra-patient HBV genetic diversity, reverse hybridization detects only known genotype-specific mutations. To resolve these limitations, we developed an Oxford Nanopore Technology (ONT)-based HBV diagnostic sequencing protocol suitable for clinical virology that yields both complete genome sequences and extensive intra-patient HBV diversity data. Specifically, the protocol involves tiling-based PCR amplification of HBV sequences, library preparation using the ONT Rapid Barcoding Kit (Oxford nanopore Technologies, Oxford, OX4 4DQ, UK), ONT GridION sequencing, genotyping using genome detective software v1.132/1.133, a recombination analysis using jpHMM (26 October 2011 version) and RDP5.61 software, and drug resistance profiling using Geno2pheno v2.0 software. We prove the utility of our protocol by efficiently generating and characterizing high-quality near full-length HBV genomes from 148 residual diagnostic samples from HBV-infected patients in the Western Cape province of South Africa, providing valuable insights into the genetic diversity and epidemiology of HBV in this region of the world.

BlaGES-producing ST654 comprises a quarter of all carbapenem-resistant Pseudomonas aeruginosa in blood isolates from 15 hospitals.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) are of major clinical concern. We analyzed 85 P. aeruginosa blood isolates non-susceptible to carbapenems collected during 2021-2023 from 15 medical centers in Israel. We aimed to determine the prevalence of high-risk clones, examine clonality, test antibiotic susceptibility, and assess the presence of acquired resistance genes, including carbapenemases. Whole-genome sequencing was performed using Illumina sequencing technology. Susceptibly was determined using the broth microdilution method. In the entire sample, 43.5% were high-risk clones. A main clade (27.1% of isolates) found in multiple hospitals comprised 19 isolates belonging to the high-risk ST654 clone and four closely related isolates. The isolates in this main clade harbored a broad set of resistance genes, including GES-type genes, and 91% had a mutated outer membrane protein (OprD). Isolates in the main clade were uniformly tobramycin (TOB) resistant and 83% were ceftolozane/tazobactam resistant. In the entire sample, we found high resistance to most antipseudomonal agents, including new beta-lactam/beta-lactamase inhibitor combinations. No uniform susceptibility to an antipseudomonal agent was found. Carbapenemases were carried by 9.4% of isolates (5.9% blaGES-5 and 3.5% blaNDM-1) and oprD was mutated in 67% of isolates. Thus, the epidemiology of CRPA is explained by a combination of clonal expansion of a dominant high-risk clade and sporadic occurrence of mutated strains. Our findings highlight the importance of susceptibility testing using a wide panel of antibiotics when CRPA is detected. Prevention measures tracking and controlling emerging high-risk clades and clones are crucial to limit the spread of CRPA.

Clinical Outcomes and Molecular Characteristics of Bacteroides fragilis Infections.

Bacteroides fragilis is the most common opportunistic anaerobic pathogen. In the absence of appropriate antimicrobial therapy, mortality rates associated with B. fragilis group infections can reach as high as 50%. Therefore, we aimed to elucidate the clinical characteristics and outcomes of B. fragilis infections and the molecular genetic characteristics of B. fragilis isolates. Forty B. fragilis clinical isolates were collected at Hanyang University Hospital between January 2022 and December 2023. Antimicrobial susceptibility was tested using the agar dilution method. Whole-genome sequencing was conducted using the Illumina platform (Illumina, San Diego, CA, USA). Various multilocus sequence types of B. fragilis were identified, including ST149 (N=4), ST11 (N=4), ST1 (N=3), ST21 (N=2), and ST157 (N=1). The insertion sequence (IS) IS1187, located upstream of cfiA, was associated with high-level carbapenem resistance in the ST157 isolate. B. fragilis toxin genes (bft ) were identified in 30% of isolates. The most common comorbidities were diabetes mellitus (26.5%) and non-metastatic cancer (23.5%). Five patients (14.7%) died within 30 days, and two (5.9%) deaths were directly attributable to B. fragilis infection. The emergence of high-level MIC carbapenem-resistant B. fragilis ST157 has led to caution in the presence of B. fragilis infections.

Large-Scale Pharmacogenomics Analysis of Patients With Cancer Within the 100,000 Genomes Project Combining Whole-Genome Sequencing and Medical Records to Inform Clinical Practice.

As part of the 100,000 Genomes Project, we set out to assess the potential viability and clinical impact of reporting genetic variants associated with drug-induced toxicity for patients with cancer recruited for whole-genome sequencing (WGS) as part of a genomic medicine service. Germline WGS from 76,805 participants was analyzed for pharmacogenetic (PGx) variants in four genes (DPYD, NUDT15, TPMT, UGT1A1) associated with toxicity induced by five drugs used in cancer treatment (capecitabine, fluorouracil, mercaptopurine, thioguanine, irinotecan). Linking genomic data with prescribing and hospital incidence records, a phenome-wide association study (PheWAS) was performed to identify whether phenotypes indicative of adverse drug reactions (ADRs) were enriched in drug-exposed individuals with the relevant PGx variants. In a subset of 7,081 patients with cancer, DPYD variants were reported back to clinicians and outcomes were collected. We identified clinically relevant PGx variants across the four genes in 62.7% of participants in our cohort. Extending this to annual prescription numbers in England for the drugs affected by these PGx variants, approximately 14,540 patients per year could potentially benefit from a reduced dose or alternative drug to reduce the risk of ADRs. Validating PGx associations in a real-world data set, we found a significant association between PGx variants in DPYD and toxicity-related phenotypes in patients treated with capecitabine or fluorouracil. Reported DPYD variants were deemed informative for clinical decision making in a majority of cases. Reporting PGx variants from germline WGS relevant to patients with cancer alongside primary findings related to their cancer can be clinically informative, informing prescribing to reduce the risk of ADRs. Extending the range of actionable variants to those found in patients of non-European ancestry is important and will extend the potential clinical impact.

Analysis of population heterogeneity in CHO cells by genome-wide DNA methylation analysis and by multi-modal single-cell sequencing

CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic landscape and heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures.