Analysis Of Whole Genome Sequencing Data Research Articles

Association mapping with a diverse population of Puccinia graminis f. sp. tritici identified avirulence loci interacting with the barley Rpg1 stem rust resistance gene

BackgroundWheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is an important disease of barley and wheat. A diverse sexual Pgt population from the Pacific Northwest (PNW) region of the US contains a high proportion of individuals with virulence on the barley stem rust resistance (R) gene, Rpg1. However, the evolutionary mechanisms of this virulence on Rpg1 are mysterious considering that Rpg1 had not been deployed in the region and the gene had remained remarkably durable in the Midwestern US and prairie provinces of Canada.Methods and resultsTo identify AvrRpg1 effectors, genome wide association studies (GWAS) were performed using 113 Pgt isolates collected from the PNW (n = 89 isolates) and Midwest (n = 24 isolates) regions of the US. Disease phenotype data were generated on two barley lines Morex and the Golden Promise transgenic (H228.2c) that carry the Rpg1 gene. Genotype data was generated by whole genome sequencing (WGS) of 96 isolates (PNW = 89 isolates and Midwest = 7 isolates) and RNA sequencing (RNAseq) data from 17 Midwestern isolates. Utilizing ~1.2 million SNPs generated from WGS and phenotype data (n = 96 isolates) on the transgenic line H228.2c, 53 marker trait associations (MTAs) were identified. Utilizing ~140 K common SNPs generated from combined analysis of WGS and RNAseq data, two significant MTAs were identified using the cv Morex phenotyping data. The 55 MTAs defined two distinct avirulence loci, on supercontig 2.30 and supercontig 2.11 of the Pgt reference genome of Pgt isolate CRL 75-36-700-3. The major avirulence locus designated AvrRpg1A was identified with the GWAS using both barley lines and was delimited to a 35 kb interval on supercontig 2.30 containing four candidate genes (PGTG_10878, PGTG_10884, PGTG_10885, and PGTG_10886). The minor avirulence locus designated AvrRpg1B identified with cv Morex contained a single candidate gene (PGTG_05433). AvrRpg1A haplotype analysis provided strong evidence that a dominant avirulence gene underlies the locus.ConclusionsThe association analysis identified strong candidate AvrRpg1 genes. Further analysis to validate the AvrRpg1 genes will fill knowledge gaps in our understanding of rust effector biology and the evolution and mechanism/s of Pgt virulence on Rpg1.

Genome analysis of Salmonella enterica serovar enteritis strains isolated from poultry and humans in Burkina Faso.

Whole-genome sequencing (WGS) was used to characterize four Salmonella enterica Enteritidis isolates from poultry (n=2) and human (n=2) from Ouagadougou, Burkina Faso. Antimicrobial resistance genes, chromosomal mutations, and mobile genetic elements were identified by analysis of WGS data using sequence homology.

RSV-GenoScan: An automated pipeline for whole-genome human respiratory syncytial virus (RSV) sequence analysis

BackgroundAdvances in high-throughput sequencing (HTS) technologies and reductions in sequencing costs have revolutionised the study of genomics and molecular biology by making whole-genome sequencing (WGS) accessible to many laboratories. However, the analysis of WGS data requires significant computational effort, which is the major drawback in implementing WGS as a routine laboratory technique. ObjectiveAutomated pipelines have been developed to overcome this issue, but they do not exist for all organisms. This is the case for human respiratory syncytial virus (RSV), which is a leading cause of lower respiratory tract infections in infants, the elderly, and immunocompromised adults. ResultsWe present RSV-GenoScan, a fast and easy-to-use pipeline for WGS analysis of RSV generated by HTS on Illumina or Nanopore platforms. RSV-GenoScan automates the WGS analysis steps directly from the raw sequence data. The pipeline filters the sequence data, maps the reads to the RSV reference genomes, generates a consensus sequence, identifies the RSV subgroup, and lists amino acid mutations, insertions and deletions in the F and G viral genes. This enables the rapid identification of mutations in these coding genes that are known to confer resistance to monoclonal antibodies. AvailabilityRSV-GenoScan is freely available at https://github.com/AlexandreD-bio/RSV-GenoScan.

Abstract 3887: The origins and clinical impact of extrachromosomal DNA across 39 cancers

Abstract Extrachromosomal DNA (ecDNA) is emerging as a major contributor to cancer drug resistance and poor outcome in many cancer types. To understand the underlying genetic and mutational processes associated with ecDNA formation and presence, we conducted an analysis of whole-genome sequencing data from 14,778 cancer patients representing 39 tumor types from the Genomics England cohort. Our findings revealed focal amplifications driven by ecDNA in 17.1% of tumor samples, exhibiting varying frequencies across tissues. Notably, liposarcomas (55.4%) and HER2+ breast cancers (46.4%) displayed higher rates compared to lower rates found in pancreatic adenocarcinomas (2%). In addition to oncogenic amplifications such as HER2 derived from ecDNA during cancer evolution, ecDNAs that did not contain oncogenes were enriched for immunomodulatory genes (e.g., CXCL6, LRRC32, and HLA-G). Immunomodulatory ecDNAs exhibited reduced T cell infiltration. In the context of intrinsic and environmental mutational processes, deamination clock-like, tobacco smoking, and APOBEC cytidine deaminase signatures positively correlated with ecDNA presence. Conversely, mismatch repair deficiency (MMRd) and POLD1/POLE deficiency signatures exhibited a negative correlation. Mutational processes associated with tobacco exposure, UV light, and spontaneous deamination primarily occurred before ecDNA formation, while signatures of homologous recombination repair deficiency tended to manifest post-ecDNA formation.Notably, ecDNA was enriched at metastatic sites and significantly associated with shorter overall survival in multivariable analysis (HR 1.31, 95% CI 1.17-1.47), underscoring its clinical relevance. This study enhances our understanding of the mutational processes and tissue contexts that shape the ecDNA landscape in human cancer and highlights its implications for clinical outcomes. Citation Format: Chris Bailey, Oriol Pich, Kerstin Thol, Jens Luebeck, Georgia Stavrou, Thomas B. Watkins, Natasha E. Weiser, Wei-Ting Lu, Jeanette Kittel, Robert Bentham, Natasha Sharma, Roberto Salgado, Dave Moore, Maria Litovchenko, King L. Hung, Alex J. Cornish, Bhargavi Dameracharla, Serena Nik-Zainal, Vineet Bafna, Howard Chang, Nicholas McGranahan, Nnennaya Kanu, Genomics England Consortium, Paul S. Mischel, Mariam Jamal-Hanjani, Charles Swanton. The origins and clinical impact of extrachromosomal DNA across 39 cancers [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 3887.

Phenotypic and genotypic characterization of Neisseria gonorrhoeae isolates among individuals at high risk for sexually transmitted diseases in Zurich, Switzerland.

While ceftriaxone resistance remains scarce in Switzerland, global Neisseria gonorrhoeae (NG) antimicrobial resistance poses an urgent threat. This study describes clinical characteristics in MSM (men who have sex with men) diagnosed with NG infection and analyses NG resistance by phenotypic and genotypic means. Data of MSM enrolled in three clinical cohorts with a positive polymerase chain reaction test (PCR) for NG were analysed between January 2019 and December 2021 and linked with antibiotic susceptibility testing. Bacterial isolates were subjected to whole genome sequencing (WGS). Of 142 participants, 141 (99%) were MSM and 118 (84%) living with HIV. Participants were treated with ceftriaxone (N = 79), azithromycin (N = 2), or a combination of both (N = 61). No clinical or microbiological failures were observed. From 182 positive PCR samples taken, 23 were available for detailed analysis. Based on minimal inhibitory concentrations (MICs), all isolates were susceptible to ceftriaxone, gentamicin, cefixime, cefpodoxime, ertapenem, zoliflodacin, and spectinomycin. Resistance to azithromycin, tetracyclines and ciprofloxacin was observed in 10 (43%), 23 (100%) and 11 (48%) of the cases, respectively. Analysis of WGS data revealed combinations of resistance determinants that matched with the corresponding phenotypic resistance pattern of each isolate. Among the MSM diagnosed with NG mainly acquired in Switzerland, ceftriaxone MICs were low for a subset of bacterial isolates studied and no treatment failures were observed. For azithromycin, high occurrences of in vitro resistance were found. Gentamicin, cefixime, cefpodoxime, ertapenem, spectinomycin, and zoliflodacin displayed excellent in vitro activity against the 23 isolates underscoring their potential as alternative agents to ceftriaxone.

Streptococcus pneumoniae serotypes 8 and 22F causing invasive pneumococcal disease in the Czech Republic in 2014-2020: whole genome sequencing (WGS) analysis.

An analysis is presented of whole genome data of Streptococcus pneumoniae serotypes 8 and 22F isolated in the Czech Republic from invasive pneumococcal disease (IPD) in 2014-2020. New multivalent pneumococcal conjugate vaccines (PCVs) are effective against these serotypes. Recently, serotypes 8 and 22F have been among the leading causes of IPD in the Czech Republic.S. pneumoniae isolates from the Czech Republic were compared with those of the same serotypes recovered in other countries in the same period and available in the international database PubMLST. Isolates from IPD of serotypes 8 (22 isolates) and 22F (21 isolates) recovered in the Czech Republic in 2014-2020 were subjected to whole genome sequencing (WGS). The genomes were analysed and compared using the international database PubMLST. Most of the studied Czech serotype 8 isolates belong to two main subpopulations. The first subpopulation, dominated by ST-53 isolates, is part of a highly abundant group of genetically close European and non-European isolates that are clearly separated on the phylogenetic network. The second subpopulation of Czech serotype 8 isolates (dominated by ST-404) is more genetically variable and forms a separate lineage on the global phylogenetic network, with no other European isolates. Czech isolates of serotype 22F are a homogeneous population with a clear predominance of ST-433, which belongs to a genetically close European population. The analysis of WGS data of IPD isolates of serotypes 8 and 22F provided a detailed insight into the genetic relationships between the Czech populations of these serotypes. It also allowed comparison of the Czech populations with the matched populations from other European and non-European countries. The obtained results add to the body of knowledge about the spread of genetic lineages causing IPD in the Czech Republic in the post-vaccination period and provide a basis for considering whether the use of the new multivalent PCVs in the Czech Republic would be beneficial.

Mapping restricted introgression across the genomes of admixed indigenous African cattle breeds

BackgroundThe genomes of indigenous African cattle are composed of components with Middle Eastern (taurine) and South Asian (indicine) origins, providing a valuable model to study hybridization and to identify genetic barriers to gene flow. In this study, we analysed indigenous African cattle breeds as models of hybrid zones, considering taurine and indicine samples as ancestors. In a genomic cline analysis of whole-genome sequence data, we considered over 8 million variants from 144 animals, which allows for fine-mapping of potential genomic incompatibilities at high resolution across the genome.ResultsWe identified several thousand variants that had significantly steep clines (‘SCV’) across the whole genome, indicating restricted introgression. Some of the SCV were clustered into extended regions, with the longest on chromosome 7, spanning 725 kb and including 27 genes. We found that variants with a high phenotypic impact (e.g. indels, intra-genic and missense variants) likely represent greater genetic barriers to gene flow. Furthermore, our findings provide evidence that a large proportion of breed differentiation in African cattle could be linked to genomic incompatibilities and reproductive isolation. Functional evaluation of genes with SCV suggest that mitonuclear incompatibilities and genes associated with fitness (e.g. resistance to paratuberculosis) could account for restricted gene flow in indigenous African cattle.ConclusionsTo our knowledge, this is the first time genomic cline analysis has been applied to identify restricted introgression in the genomes of indigenous African cattle and the results provide extended insights into mechanisms (e.g. genomic incompatibilities) contributing to hybrid differentiation. These results have important implications for our understanding of genetic incompatibilities and reproductive isolation and provide important insights into the impact of cross-breeding cattle with the aim of producing offspring that are both hardy and productive.

Whole-Genome Sequencing-Based Profiling of Antimicrobial Resistance Genes and Core-Genome Multilocus Sequence Typing of Campylobacter jejuni from Different Sources in Lithuania.

Campylobacter jejuni is known as one of the main causative agents of gastroenteritis in humans worldwide, and the rise of antimicrobial resistance (AMR) in Campylobacter is a growing public health challenge of special concern. Whole-genome sequencing (WGS) was used to characterize genetic determinants of AMR in 53 C. jejuni isolates from dairy cattle, broiler products, wild birds, and humans in Lithuania. The WGS-based study revealed 26 C. jejuni AMR markers that conferred resistance to various antimicrobials. Genetic markers associated with resistance to beta-lactamases, tetracycline, and aminoglycosides were found in 79.3%, 28.3%, and 9.4% of C. jejuni isolates, respectively. Additionally, genetic markers associated with multidrug resistance (MDR) were found in 90.6% of C. jejuni isolates. The WGS data analysis revealed that a common mutation in the quinolone resistance-determining region (QRDR) was R285K (854G > A) at 86.8%, followed by A312T (934G > A) at 83% and T86I (257C > T) at 71.7%. The phenotypic resistance analysis performed with the agar dilution method revealed that ciprofloxacin (CIP) (90.6%), ceftriaxone (CRO) (67.9%), and tetracycline (TET) (45.3%) were the predominant AMR patterns. MDR was detected in 41.5% (22/53) of the isolates tested. Fifty-seven virulence genes were identified in all C. jejuni isolates; most of these genes were associated with motility (n = 28) and chemotaxis (n = 10). Additionally, all C. jejuni isolates harbored virulence genes related to adhesion, invasion, LOS, LPS, CPS, transportation, and CDT. In total, 16 sequence types (STs) and 11 clonal complexes (CC) were identified based on core-genome MLST (cgMLST) analysis. The data analysis revealed distinct diversity depending on phenotypic and genotypic antimicrobial resistance of C. jejuni.

Genetic determinants underlying the progressive phenotype of β-lactam/β-lactamase inhibitor resistance in Escherichia coli.

Currently, whole-genome sequencing (WGS) data have not shown strong concordance with Escherichia coli susceptibility profiles to the commonly used β-lactam/β-lactamase inhibitor (BL/BLI) combinations: ampicillin-sulbactam (SAM), amoxicillin-clavulanate (AMC), and piperacillin-tazobactam (TZP). Progressive resistance to these BL/BLIs in the absence of cephalosporin resistance, also known as extended-spectrum resistance to BL/BLI (ESRI), has been suggested to primarily result from increased copy numbers of blaTEM variants, which is not routinely assessed in WGS data. We sought to determine whether addition of gene amplification could improve genotype-phenotype associations through WGS analysis of 147 E. coli bacteremia isolates with increasing categories of BL/BLI non-susceptibility ranging from ampicillin (AMP) susceptibility to being fully resistant to all three BL/BLIs. Consistent with a key role of blaTEM in ESRI, 112/134 strains (84%) with at least ampicillin non-susceptibility encoded blaTEM. Evidence of blaTEM amplification (i.e., blaTEM gene copy number estimates > 2×) was present in 40/112 (36%) strains. There were positive correlations between blaTEM copy numbers with minimum inhibitory concentrations of AMC and TZP (P < 0.05) but not for SAM (P = 0.09). The diversity of β-lactam resistance mechanisms, including non-ceftriaxone hydrolyzing blaCTX-M variants, blaOXA-1, and ampC and blaTEM strong promoter mutations, was greater in AMC- and TZP-non-susceptible strains but rarely observed within SAM- and AMP-non-susceptible isolates. Our study indicates that comprehensive analysis of WGS data, including β-lactamase-encoding gene amplification, can help categorize E. coli with AMC or TZP non-susceptibility but that discerning the transition from SAM susceptibility to SAM non-susceptibility using genetic data requires further refinement. IMPORTANCE The increased feasibility of whole-genome sequencing has generated significant interest in using such molecular diagnostic approaches to characterize difficult-to-treat, antimicrobial-resistant (AMR) infections. Nevertheless, there are current limitations in the accurate prediction of AMR phenotypes based on existing AMR gene database approaches, which primarily correlate a phenotype with the presence/absence of a single AMR gene. Our study utilized a large cohort of cephalosporin-susceptible Escherichia coli bacteremia samples to determine how increasing the dosage of narrow-spectrum β-lactamase-encoding genes in conjunction with other diverse β-lactam/β-lactamase inhibitor (BL/BLI) genetic determinants contributes to progressively more severe BL/BLI phenotypes. We were able to characterize the complexity of the genetic mechanisms underlying progressive BL/BLI resistance including the critical role of β-lactamase encoding gene amplification. For the diverse array of AMR phenotypes with complex mechanisms involving multiple genomic factors, our study provides an example of how composite risk scores may improve understanding of AMR genotype/phenotype correlations.

ABCA4 c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease.

Introduction: Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 (ABCA4) gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported ABCA4 variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Methods: Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both in silico and in vitro. The in silico approach was based on the deep-learning tool SpliceAI. For the in vitro approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing ABCA4 exons 46 to 48. Results: Through the analysis of WGS data, we identified two candidate variants in ABCA4 in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in trans. For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases. Conclusion: A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

Use of genome sequencing to hunt for cryptic second-hit variants: analysis of 31 cases recruited to the 100 000 Genomes Project

BackgroundCurrent clinical testing methods used to uncover the genetic basis of rare disease have inherent limitations, which can lead to causative pathogenic variants being missed. Within the rare disease arm...

Selection signatures and population dynamics of transposable elements in lima bean

The domestication process in lima bean (Phaseolus lunatus L.) involves two independent events, within the Mesoamerican and Andean gene pools. This makes lima bean an excellent model to understand convergent evolution. The mechanisms of adaptation followed by Mesoamerican and Andean landraces are largely unknown. Genes related to these adaptations can be selected by identification of selective sweeps within gene pools. Previous genetic analyses in lima bean have relied on Single Nucleotide Polymorphism (SNP) loci, and have ignored transposable elements (TEs). Here we show the analysis of whole-genome sequencing data from 61 lima bean accessions to characterize a genomic variation database including TEs and SNPs, to associate selective sweeps with variable TEs and to predict candidate domestication genes. A small percentage of genes under selection are shared among gene pools, suggesting that domestication followed different genetic avenues in both gene pools. About 75% of TEs are located close to genes, which shows their potential to affect gene functions. The genetic structure inferred from variable TEs is consistent with that obtained from SNP markers, suggesting that TE dynamics can be related to the demographic history of wild and domesticated lima bean and its adaptive processes, in particular selection processes during domestication.

Large-Scale Whole Genome Sequence Analysis of >22,000 Subjects Provides no Evidence of FMR1 Premutation Allele Involvement in Autism Spectrum Disorder.

Expansion of a CGG repeat in the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene on the X chromosome is the cause of Fragile X Syndrome (FXS). The repeat length of unaffected individuals varies between 5-40 repeats, whereas >200 repeats are observed in cases of FXS. The intermediate range between 55-200 repeats is considered the premutation range and is observed in roughly 1:300 females and 1:900 males in the general population. With the availability of large-scale whole genome sequence (WGS) data and the development of computational tools to detect repeat expansions, we systematically examined the role of FMR1 premutation alleles in autism spectrum disorder (ASD) susceptibility, assess the prevalence, and consider the allelic stability between parents and offspring. We analyzed the WGS data of 22,053 subjects, including 32 FXS positive controls, 1359 population controls, and 5467 ASD families. We observed no FMR1 full mutation range repeats among the ASD parent-offspring families but identified 180 family members with premutation range alleles, which represents a higher prevalence compared to the independent WGS control sample and previous reports in the literature. A sex-specific analysis between probands and unaffected siblings did not reveal a significant increase in the burden of premutation alleles in either males or females with ASD. PCR validation, however, suggests an overestimation of the frequency of FMR1 premutation range alleles through computational analysis of WGS data. Overall, we show the utility of large-scale repeat expansion screening in WGS data and conclude that there is no apparent evidence of FMR1 premutation alleles contributing to ASD susceptibility.

Genomic Characterization of Fecal Escherichia coli Isolates with Reduced Susceptibility to Beta-Lactam Antimicrobials from Wild Hogs and Coyotes.

This study was carried out to determine the antimicrobial resistance (AMR) genes and mobile genetic elements of 16 Escherichia coli isolates-with reduced susceptibility to ceftazidime and imipenem-that were recovered from the fecal samples of coyotes and wild hogs from West Texas, USA. Whole-genome sequencing data analyses revealed distinct isolates with a unique sequence type and serotype designation. Among 16 isolates, 4 isolates were multidrug resistant, and 5 isolates harbored at least 1 beta-lactamase gene (blaCMY-2, blaCTX-M-55, or blaCTX-M-27) that confers resistance to beta-lactam antimicrobials. Several isolates carried genes conferring resistance to tetracyclines (tet(A), tet(B), and tet(C)), aminoglycosides (aac(3)-IId, ant(3″)-Ia, aph(3')-Ia, aph(3″)-lb, aadA5, and aph(6)-ld), sulfonamides (sul1, sul2, and sul3), amphenicol (floR), trimethoprim (dfrA1 and dfrA17), and macrolide, lincosamide, and streptogramin B (MLSB) agents (Inu(F), erm(B), and mph(A)). Nine isolates showed chromosomal mutations in the promoter region G of ampC beta-lactamase gene, while three isolates showed mutations in gyrA, parC, and parE quinolone resistance-determining regions, which confer resistance to quinolones. We also detected seven incompatibility plasmid groups, with incF being the most common. Different types of virulence genes were detected, including those that enhance bacterial fitness and pathogenicity. One blaCMY-2 positive isolate (O8:H28) from a wild hog was also a Shiga toxin-producing E. coli and was a carrier of the stx2A virulence toxin subtype. We report the detection of blaCMY-2, blaCTX-M-55, and blaCTX-M-27 beta-lactamase genes in E. coli from coyotes for the first time. This study demonstrates the importance of wildlife as reservoirs of important multi-drug-resistant bacteria and provides information for future comparative genomic analysis with the limited literature on antimicrobial resistance dynamics in wildlife such as coyotes.

Mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (β-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

Genome Investigation of Brucella melitensis Strains Isolated From Brucellosis Patients in Kuwait

Background: Human brucellosis is present on all inhabited continents with high prevalence in many areas of the world, including Kuwait and the Middle East. To implement proper control measures, the identification and characterization of Brucella species and genotypes are required through a reliable and rapid subtyping method. In previous studies, whole-genome sequencing (WGS) has shown its potential as an epidemiological typing tool. Using WGS data, this study aimed to identify the species, phage sequences, putative antibiotic resistance genes, virulence factors, and genotypes of Brucella melitensis strains isolated from patients in Kuwait and other countries. Methods: Five B. melitensis isolates of Kuwaiti origin and 31 other isolates of B. melitensis originating from 28 countries were analyzed using whole genome-based approaches for genotypic identification and typing. In-silico techniques were used to identify the sequences for phages, antibiotic resistance genes, virulence factors, and genotypes using multilocus sequence typing (MLST) and whole-genome single-nucleotide polymorphism (wgSNP). Results: The analysis of WGS data demonstrated that all five Kuwaiti isolates belonged to the non-vaccine strains of B. melitensis. Furthermore, the data represented the presence of two phage sequences, two antibiotic resistance genes, and 51 virulence factors in Kuwaiti isolates. Eventually, the genotypes of all isolates were identified based on MLST and wgSNP analysis, and wgSNP analysis suggested the possible areas/countries of origin of Kuwaiti isolates. Conclusion: WGS data can be used to characterize Brucella isolates, and molecular techniques can be applied in-silico to rapidly identify and classify Brucella into species and genotypes and trace the possible origin of the isolates.

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for “correcting” variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Abstract A017: Molecular analysis of matched PIN, invasive prostate cancer, and adjacent normal prostate tissue samples reveal distinct transcriptional signatures and clonal relationships

Abstract Background: Prostate cancer (PCa) is highly heterogeneous, and while some tumors remain indolent throughout a patient's lifetime, others are highly aggressive, leading to significant morbidity and death. The current methods for PCa diagnosis and prognosis are highly suboptimal, resulting in over-diagnosis and overtreatment of many clinically insignificant tumors. To understand the nature of PCa heterogeneity, we conducted comprehensive genomic analyses on matched samples of adjacent normal prostate tissue, prostate intraepithelial neoplasia (PIN), and invasive PCa. Methods: To investigate the changes cells undergo during PCa development and progression, we analyzed genomic and transcriptomic alterations in 92 adjacent normal, 80 PIN, and 99 PCa samples from 34 patients. For each sample type, both epithelial and stromal components were analyzed separately using laser capture microdissection of formalin fixed paraffin embedded tissue. Transcriptomic analysis was performed using smart3-SEQ and copy number analysis was performed by low-pass whole genome sequencing (WGS). The samples were analyzed as three separate data sets. Results: We studied the spectrum of molecular changes present in both epithelial and stromal samples. A negative-binomial regression model was used to identify differentially expressed genes between adjacent normal prostate tissue and invasive PCa in epithelium and stroma separately using the discovery data set. The resulting gene lists were used to perform non-negative matrix factorization clustering of all samples in the three datasets separately. This analysis identified two epithelial and two stromal clusters with distinct RNA expression profiles in all three data sets. For invasive PCa samples, all except three epithelial samples and five stromal samples clustered in the ‘invasive-like’ clusters. Similarly for adjacent normal samples, all except three of the epithelial and six of the stromal samples clustered as ‘normal-like’ across the three datasets. For PIN samples, 54% of epithelial and 62% of stromal samples classified as ‘normal-like’. We performed phylogenetic analysis of WGS data and identified PIN lesions with and without clear clonal relation to invasive PCa lesions. Recurrent copy number variations were identified in both PIN and PCa lesions. Conclusion. We identified two distinct epithelial and two distinct stromal expression clusters with ‘invasive-like’ and ‘normal-like’ signatures. WGS analysis revealed recurrent copy number alterations in PIN and PCa lesions, and evidence of clonal relationships between PIN and invasive PCa. These studies provide new insight into PIN biology and the relationship between PIN and invasive PCa. Citation Format: Siri H. Strand, Okyaz Eminaga, Sujay Vennam, Chunfang Zhu, Jason Wang, Sushama Varma, Rosalie Nolley, Christian Kunder, Jonathan Pollack, Andreas Roeder, Karina D. Sorensen, James D. Brooks, Robert B. West. Molecular analysis of matched PIN, invasive prostate cancer, and adjacent normal prostate tissue samples reveal distinct transcriptional signatures and clonal relationships [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A017.

Foodborne Outbreak of Extended Spectrum Beta-lactamase Producing Shigella sonnei Associated with Contaminated Spring Onions in the United Kingdom

Globalization of the food supply chain has created conditions favorable for emergence and spread of multidrug-resistant (MDR) foodborne pathogens. In November 2021, the UK Health Security Agency detected an outbreak of 17 cases infected with the same strain of MDR extended spectrum beta-lactamase (ESBL)-producing Shigella sonnei. Phylogenetic analysis of whole-genome sequencing data revealed the outbreak was closely related to strains of S. sonnei isolated from travelers returning to the UK from Egypt. None of the outbreak cases reported travel and all 17 cases reported eating food from a restaurant/food outlet in the week prior to symptom onset, of which 11/17 (64.7%) ate at branches of the same national restaurant franchise. All 17 cases were adults and 14/17 (82.4%) were female. Ingredient-level analyses of the meals consumed by the cases identified spring onions as the common ingredient. Food chain investigations revealed that the spring onions served at the implicated restaurants could be traced back to a single Egyptian producer. The foodborne transmission of ESBL-producing bacteria is an emerging global health concern, and concerted action from all stakeholders is required to ensure an effective response to mitigate the risks to public health.

Abstract 2305: Ultra-sensitive detection of circulating tumor DNA by whole-genome sequencing of blood samples from locally advanced rectal cancer patients receiving neoadjuvant therapy and enrolled in watch-and-wait strategies for organ preservation

Abstract Background. We hypothesized that circulating tumor DNA (ctDNA) can be used as a prognostic biomarker, improve the assessment of response and enhance detection of minimal residual disease in patients with locally advanced rectal cancer (LARC) treated with neoadjuvant therapy (NAT). Methods. We analyzed data from 31 LARC patients treated at Memorial Sloan Kettering as part of the Organ Preservation in Rectal Adenocarcinoma (OPRA) phase II clinical trial. Patients had stage II or III rectal adenocarcinoma and received NAT including chemoradiation and chemotherapy. Patients without a clinical complete response (cCR) underwent surgical resection, while patients with a cCR were enrolled in a watch-and-wait protocol for organ preservation. Complete response (CR) after NAT was defined as either pathological complete response or a cCR sustained for ≥2 years. Disease-free survival (DFS) was measured from the start of NAT. Median follow-up was 5.41 years [range 2.96-8.38]. ctDNA analyses were performed using the C2i Genomics platform. A patient-specific molecular profile was created by performing whole-genome sequencing (WGS) of their tumor and matched normal DNA (40x coverage). WGS (20x coverage) was performed on plasma samples collected at baseline (before NAT), interval evaluation (halfway through NAT), re-staging evaluation (8 weeks after NAT) and follow-up (3-6 months after NAT). Results. Tumor was detected in plasma samples from 24/25 patients at baseline (96% sensitivity). The tumor fraction (TF) levels detected at baseline separated responders from non-responders (median TF 6.2e-4 vs 1.4e-3; p=0.055). Tumor detection at interval was associated with a lower rate of CR (25% vs. 75%, p=0.0095) and shorter time to recurrence (58.3% vs. 94.1% 3-year DFS, p=0.02). Tumor detection at follow-up was associated with a higher rate of recurrence (p=0.037) and tumor was detected at follow-up for all 5/5 patients who developed recurrence. Overall TF dynamics showed clearance of ctDNA down to the non-detection level throughout treatment in patients with a CR, while non-responders exhibited non-decreasing and often increasing estimates of ctDNA burden. Analysis of tissue WGS data identified multiple patients with colibactin associated mutational signatures, which provides additional insights into their cancer etiology. Conclusions. The WGS-based approach for ctDNA analysis exhibited very high sensitivity for detection at baseline. TF across multiple time points separated responders from non-responders, suggesting potential value as a prognostic marker. Detection of ctDNA at follow-up for all patients who recurred is indicative of potential clinical utility for treatment de-escalation in the context of organ preservation strategies. Citation Format: Francisco Sanchez-Vega, Chin-Tung Chen, Danielle Afterman, Dana Omer, Madison Darmofal, Ino de Bruijn, Walid K. Chatila, Matthew Drescher, Grittney Tam, Tomer Lauterman, Maja Kuzman, Santiago Gonzalez, Dunja Glavas, James Samdbeck, Dillon Maloney, Jurica Levatic, Sunil Deochand, Michael Yahalom, Ryan Ptashkin, Iman Tavassoly, Zohar Donenhirsh, Eric White, Ravi Kandasamy, Ury Alon, Michael F. Berger, Brian Loomis, Paz Polak, Boris Oklander, Asaf Zviran, Julio Garcia-Aguilar. Ultra-sensitive detection of circulating tumor DNA by whole-genome sequencing of blood samples from locally advanced rectal cancer patients receiving neoadjuvant therapy and enrolled in watch-and-wait strategies for organ preservation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2305.