Whether Whole-genome Sequencing Research Articles

Whole genome sequencing in families with oligodontia

AbstractBackground/ObjectivesTooth agenesis (TA) is among the most common malformations in humans. Although several causative mutations have been described, the genetic cause often remains elusive. Here, we test whether whole genome sequencing (WGS) could bridge this diagnostic gap.MethodsIn four families with TA, we assessed the dental phenotype using the Tooth Agenesis Code after intraoral examination and radiographic and photographic documentation. We performed WGS of index patients and subsequent segregation analysis.ResultsWe identified two variants of uncertain significance (a potential splice variant in PTH1R, and a 2.1 kb deletion abrogating a non‐coding element in FGF7) and three pathogenic variants: a novel frameshift in the final exon of PITX2, a novel deletion in PAX9, and a known nonsense variant in WNT10A. Notably, the FGF7 variant was found in the patient, also featuring the WNT10A variant. While mutations in PITX2 are known to cause Axenfeld‐Rieger syndrome 1 (ARS1) predominantly featuring ocular findings, accompanied by dental malformations, we found the PITX2 frameshift in a family with predominantly dental and varying ocular findings.ConclusionSevere TA predicts a genetic cause identifiable by WGS. Final exon PITX2 frameshifts can cause a predominantly dental form of ARS1.

Whole-genome analysis as a diagnostic tool for patients referred for diagnosis of Silver-Russell syndrome: a real-world study

BackgroundSilver-Russell syndrome (SRS) is an imprinting disorder characterised by prenatal and postnatal growth restriction, but its clinical features are non-specific and its differential diagnosis is broad. Known molecular causes of...

Genomic Epidemiology of Carbapenemase-Producing Enterobacterales at a Hospital System in Toronto, Ontario, Canada, 2007 to 2018.

At a hospital system (H1) in Ontario, Canada, we investigated whether whole-genome sequencing (WGS) altered initial epidemiological interpretation of carbapenemase-producing Enterobacterales (CPE) transmission. We included patients with CPE colonization/infection identified by population-based surveillance from October 2007 to August 2018 who received health care at H1 in the year before/after CPE detection. H1 reported epidemiological transmission clusters. We combined single nucleotide variant (SNV) analysis, plasmid characterization, and epidemiological data. Eighty-five patients were included. H1 identified 7 epidemiological transmission clusters, namely, A to G, involving 24/85 (28%) patients. SNV analysis confirmed transmission clusters C, D, and G and identified two additional cases belonging to cluster A. One was a travel-related case that was the likely index case (0 to 6 SNVs from other isolates); this case stayed on the same unit as the initially presumed index case 4 months prior to detection of the initially presumed index case on another unit. The second additional case occupied a room previously occupied by 5 cluster A cases. Plasmid sequence analysis excluded a case from cluster A and identified clusters E and F as possibly two parts of a single cluster. SNV analysis also identified a case without direct epidemiologic links that was 18 to 21 SNVs away from cluster B, suggesting possible undetected interhospital transmission. SNV and plasmid sequence analysis identified cases belonging to transmission clusters that conventional epidemiology missed and excluded other cases. Implementation of routine WGS to complement epidemiological transmission investigations has the potential to improve prevention and control of CPE in hospitals.

Abstract PO-093: Circulating tumor DNA (ctDNA) from peripheral blood is detectable among Ghanaian breast cancer patients

Abstract Cancer incidence is rising and mortality rates are high in Africa, where access to molecular pathology is limited. Analysis of cancer-related mutations in circulating tumor DNA (ctDNA) from cell-free DNA (cfDNA) that is shed into the bloodstream by tumor cells could be transformative to the African continent and provide new molecular insights. Using samples collected from the Ghana Breast Health Study we tested whether whole-genome sequencing (WGS) of cfDNA could detect ctDNA and identify somatic alterations that drive breast cancer. We selected 15 breast cancer patients (median age 49.5 years) with duplicated plasma samples. Pathologic grade, age, and immunohistochemical (IHC) stains for estrogen receptor (ER), progesterone receptor (PR) and HER2 were available for the majority of patients (>80%). cfDNA extraction and WGS at 30x and 0.1x was performed. ichorCNA software was used on Next Generation Sequencing (NGS) read counts to estimate the ctDNA fraction and predict copy number alteration profiles. High depth 30x cfDNA-WGS analysis showed that all 15 breast cancer patients had 1% ctDNA or greater (median[IQR] 3.96%[2.22%-8.13%]). There was high concordance between estimated ctDNA fraction using 0.1x and 30x WGS (Pearson r = 0.9). Copy number profiling showed extensive amplification and deletion of multiple chromosomal regions containing important cancer genes (such as MYC, PIK3CA, TERT, and GATA3). Of the four patients classified as HER2 positive based on IHC, two had increased ERBB2 copy number (50 and 3 copies, respectively). Our data provide evidence that ctDNA-based genomic studies are possible and ctDNA analysis could be a tool for future molecular oncology studies in Africa for cancer etiology, surveillance and clinical trials. Citation Format: Samuel T. Ahuno, Lawrence Edusei, Nicolas Titiloye, Ernest Adjei, Joe-Nat Clegg-Lamptey, Joel Yarney, Beatrice Wiafe-Addai, Baffour Awuah, Verne Vanderpuye, Maire Duggan, Seth Wiafe, Kofi Nyarko, Francis Aitpillah, Daniel Ansong, Thomas Ahearn, Alexander Kwarteng, Mustapha Abubakar, Montserrat Garcia-Closas, Gavin Ha, Jonine D. Figueroa, Paz Polak, on behalf of the Ghana Breast Health Study Team. Circulating tumor DNA (ctDNA) from peripheral blood is detectable among Ghanaian breast cancer patients [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-093.

Using Whole-Genome Sequencing to Improve Surveillance Measures: Case Study of Methicillin-Resistant Staphylococcus aureus (MRSA) in a Florida Hospital

Background: The CDC considers methicillin-resistant Staphylococcus aureus (MRSA) one of the most important hospital-acquired infections (HAIs) in the United States. However, infection control departments (ICDs) often rely on subjective data to determine whether multiple MRSA cases are a true outbreak and whether the hospital is responsible (community- vs hospital-acquired). Objective: Our objective was to determine whether whole-genome sequencing (WGS) of MRSA provided new insights into on transmission dynamics at large, inner-city hospital in Jacksonville, Florida. Methods: Over a 4-month period, MRSA samples were obtained from 44 infected patients at 3 campuses within a single hospital system. Limited nonpatient identifying information was obtained, including date of collection, campus, unit, reason for admission, and days post admission (DPA) of MRSA diagnosis. Whole-genome sequences were generated using the Illumina platform. Raw reads were processed, and genetic distances were calculated and used to identify genetically linked bacterial infections using FoxSeq version 1.0 software. Results: Based on their length of stay, 10 patients were reported by the ICD as obtaining an HAI. Three distinct “episodes” were evident in which >5 MRSA cases were observed within a 3–5-day period. Genomic analysis identified 5 clusters of linked infections: 4 clusters contained 2 patients and another contained 3. Of these clusters, only 1 contained multiple cases that were reported as HAIs; however, because these case were separated by 2 weeks, it is unlikely that they came from a source in the hospital. The results suggest that HAIs were overreported and that most MRSA present in the hospital likely came from community sources. Conclusions: WGS provided clear evidence that temporally clustered MRSA cases do not imply an outbreak is occurring. Furthermore, ongoing detection of the same community-acquired infections over several months is indicative of a shared source outside of the hospital, which could be uncovered through examination of clinical records. Considering the implications of HAIs, best approaches to combat them should include identifying their sources. As molecular surveillance approaches to infection control are rapidly becoming easier and less expensive, the methods can be used to bring objective clarity to the ICD.Funding: NoneDisclosures: Susanna L. Lamers reports salary from BioInfoExperts and contract research for the NIH, the University of California - San Francisco, and UMASS Medical School.

Genomic Epidemiology of Carbapenemase-Producing Enterobacterales (CPE) in Toronto, Canada

Objective: We investigated whether whole-genome sequencing (WGS) data altered interpretations of clonal transmission as determined by conventional epidemiology and pulsed-field gel electrophoresis (PFGE) at a tertiary-care hospital (hospital Z, HZ). Methods: We included all carbapenemase-producing Enterobacterales (CPE)–colonized or –infected patients identified via population-based surveillance from 2007 through 2018, who were admitted to HZ during and/or in the year prior to or following CPE detection. HZ reported clonal transmission clusters using epidemiology and PFGE for CPE identified at HZ or reported to HZ by other hospitals as potentially acquired at HZ. We assessed single-nucleotide polymorphism (SNP) phylogenies and case epidemiology. Results: Overall, 85 CPE-colonized or -infected patients were included: 50 were detected at HZ and 35 were detected at another local hospital but were admitted to HZ in the previous or following year. HZ reported 6 transmission clusters (Table 1). SNP analyses confirmed clusters B, C, E, and F. In cluster A, SNP analyses cast doubt on 2 of 9 cases (possibly representing plasmid transmission) but also identified 2 additional cases with isolates highly related (0–3 SNP differences) to other isolates. One case may be the index case: a travel-related case who stayed on the same unit as case 1, 4 months before case 1 detection. The second case stayed in a room previously occupied by 5 cluster A cases. In cluster D, SNP analyses found 1 additional case whose isolate was highly related (ie, 17–19 SNP differences) to other isolates. This case was identified a year before cluster D at another hospital that shares patients with HZ; however, the case’s admission to HZ was after all cluster D cases were detected and no direct epidemiologic link was identified. Conclusions: WGS data can identify cases belonging to transmission clusters that conventional epidemiologic methods missed.Funding: NoneDisclosures: Allison McGeer reports funds to her institution from Pfizer and Merck for projects for which she is the principal investigator. She also reports consulting fees from Sanofi-Pasteur, Sunovion, GSK, Pfizer, and Cidara

Reconciling the Potentially Irreconcilable? Genotypic and Phenotypic Amoxicillin-Clavulanate Resistance in Escherichia coli

Resistance to amoxicillin-clavulanate, a widely used beta-lactam/beta-lactamase inhibitor combination antibiotic, is rising globally, and yet susceptibility testing remains challenging. To test whether whole-genome sequencing (WGS) could provide a more reliable assessment of susceptibility than traditional methods, we predicted resistance from WGS for 976 Escherichia coli bloodstream infection isolates from Oxfordshire, United Kingdom, comparing against phenotypes from the BD Phoenix (calibrated against EUCAST guidelines). A total of 339/976 (35%) isolates were amoxicillin-clavulanate resistant. Predictions based solely on beta-lactamase presence/absence performed poorly (sensitivity, 23% [78/339]) but improved when genetic features associated with penicillinase hyperproduction (e.g., promoter mutations and copy number estimates) were considered (sensitivity, 82% [277/339]; P < 0.0001). Most discrepancies occurred in isolates with MICs within ±1 doubling dilution of the breakpoint. We investigated two potential causes: the phenotypic reference and the binary resistant/susceptible classification. We performed reference standard, replicated phenotyping in a random stratified subsample of 261/976 (27%) isolates using agar dilution, following both EUCAST and CLSI guidelines, which use different clavulanate concentrations. As well as disagreeing with each other, neither agar dilution phenotype aligned perfectly with genetic features. A random-effects model investigating associations between genetic features and MICs showed that some genetic features had small, variable and additive effects, resulting in variable resistance classification. Using model fixed-effects to predict MICs for the non-agar dilution isolates, predicted MICs were in essential agreement (±1 doubling dilution) with observed (BD Phoenix) MICs for 691/715 (97%) isolates. This suggests amoxicillin-clavulanate resistance in E. coli is quantitative, rather than qualitative, explaining the poorly reproducible binary (resistant/susceptible) phenotypes and suboptimal concordance between different phenotypic methods and with WGS-based predictions.

Surveillance-embedded genomic outbreak resolution of methicillin-susceptible Staphylococcus aureus in a neonatal intensive care unit

We observed an increase in methicillin-susceptible Staphylococcus aureus (MSSA) infections at a Dutch neonatal intensive care unit. Weekly neonatal MSSA carriage surveillance and cross-sectional screenings of health care workers (HCWs) were available for outbreak tracing. Traditional clustering of MSSA isolates by spa typing and Multiple-Locus Variable number tandem repeat Analysis (MLVA) suggested that nosocomial transmission had contributed to the infections. We investigated whether whole-genome sequencing (WGS) of MSSA surveillance would provide additional evidence for transmission. MSSA isolates from neonatal infections, carriage surveillance, and HCWs were subjected to WGS and bioinformatic analysis for identification and localization of high-quality single nucleotide polymorphisms, and in-depth analysis of subsets of isolates. By measuring the genetic diversity in background surveillance, we defined transmission-level relatedness and identified isolates that had been unjustly assigned to clusters based on MLVA, while spa typing was concordant but of insufficient resolution. Detailing particular subsets of isolates provided evidence that HCWs were involved in multiple outbreaks, yet it alleviated concerns about one particular HCW. The improved resolution and accuracy of genomic outbreak analyses substantially altered the view on outbreaks, along with apposite measures. Therefore, inclusion of the circulating background population has the potential to overcome current issues in genomic outbreak inference.

Use of Whole-Genome Sequencing of Adenovirus in Immunocompromised Pediatric Patients to Identify Nosocomial Transmission and Mixed-Genotype Infection.

Adenoviruses are significant pathogens for the immunocompromised, arising from primary infection or reinfection. Serotyping is insufficient to support nosocomial transmission investigations. We investigate whether whole-genome sequencing (WGS) provides clinically relevant information on transmission among patients in a pediatric tertiary hospital. We developed a target-enriched adenovirus WGS technique for clinical samples and retrospectively sequenced 107 adenovirus-positive residual diagnostic samples, including viremias (>5 × 104 copies/mL), from 37 patients collected January 2011-March 2016. Whole-genome sequencing was used to determine genotype and for phylogenetic analysis. Adenovirus sequences were recovered from 105 of 107 samples. Full genome sequences were recovered from all 20 nonspecies C samples and from 36 of 85 species C viruses, with partial genome sequences recovered from the rest. Whole-genome phylogenetic analysis suggested linkage of 3 genotype A31 cases and uncovered an unsuspected epidemiological link to an A31 infection first detected on the same ward 4 years earlier. In 9 samples from 1 patient who died, we identified a mixed genotype adenovirus infection. Adenovirus WGS from clinical samples is possible and useful for genotyping and molecular epidemiology. Whole-genome sequencing identified likely nosocomial transmission with greater resolution than conventional genotyping and distinguished between adenovirus disease due to single or multiple genotypes.

Evaluation of whole genome sequencing and software tools for drug susceptibility testing of Mycobacterium tuberculosis

ObjectivesCulture-based assays are currently the reference standard for drug susceptibility testing for Mycobacterium tuberculosis. They provide good sensitivity and specificity but are time consuming. The objective of this study was to evaluate whether whole genome sequencing (WGS), combined with software tools for data analysis, can replace routine culture-based assays for drug susceptibility testing of M. tuberculosis. MethodsM. tuberculosis cultures sent to the Finnish mycobacterial reference laboratory in 2014 (n = 211) were phenotypically tested by Mycobacteria Growth Indicator Tube (MGIT) for first-line drug susceptibilities. WGS was performed for all isolates using the Illumina MiSeq system, and data were analysed using five software tools (PhyResSE, Mykrobe Predictor, TB Profiler, TGS-TB and KvarQ). Diagnostic time and reagent costs were estimated for both methods. ResultsThe sensitivity of the five software tools to predict any resistance among strains was almost identical, ranging from 74% to 80%, and specificity was more than 95% for all software tools except for TGS-TB. The sensitivity and specificity to predict resistance to individual drugs varied considerably among the software tools. Reagent costs for MGIT and WGS were €26 and €143 per isolate respectively. Turnaround time for MGIT was 19 days (range 10–50 days) for first-line drugs, and turnaround time for WGS was estimated to be 5 days (range 3–7 days). ConclusionsWGS could be used as a prescreening assay for drug susceptibility testing with confirmation of resistant strains by MGIT. The functionality and ease of use of the software tools need to be improved.

Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing

ObjectivesPseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the...

Whole Genome Sequencing in Clinical Practice: Promise and Caution

Significant advances in DNA sequencing now permit the sequencing of an individual's whole genome for ∼$1,000. This will soon result in a paradigm shift in how we approach the diagnosis and treatment of rare and common diseases.1 The question we should carefully consider is whether whole genome sequencing (WGS) is ready for routine use in clinical practice. While one WGS sample can generate gigabytes of data, whether these data can generate meaningful information is significantly more complex. Analysis of the raw data requires specialized bioinformatics expertise and complicated software. Poor initial analysis of the raw …

A whole-genome sequencing approach to targeting Mycobacterium tuberculosis outbreak management

BackgroundEpidemiological investigations into Mycobacterium tuberculosis outbreaks use 24-locus genotyping (MIRU-VNTR typing). Where no epidemiological link can be found between patients, the importance of shared genotypes remains unclear. This issue is especially problematic and time-consuming when tracing contacts within some social groups at high tuberculosis risk, in which unwillingness to volunteer information is common. We investigated whether whole-genome sequencing (WGS) could delineate outbreaks with greater resolution than MIRU-VNTR typing has done. MethodsWe sequenced 390 M tuberculosis isolates from 254 patients from the UK Midlands (1994–2011) using Illumina technology (appendix). We estimated the expected genomic diversity between isolates within a transmission chain by measuring pairwise nucleotide differences between genomes within hosts (79 individuals with pulmonary and extrapulmonary disease, or multiple pulmonary episodes) and between hosts within 25 household outbreaks (63 individuals). We then investigated 11 MIRU-VNTR-based community clusters (168 patients, 157 transmission events) to assess whether WGS could delineate outbreaks more effectively. For each cluster we reconstructed the most plausible transmission chain based on epidemiological data collected by tuberculosis nurses, pairwise nucleotide distances, and times of diagnosis, and compared the genomic diversity across these constructed links with that within individuals and within household outbreaks. Findings109 (96%) of 114 isolates were within five SNPs of another isolate taken from the same individual or from an individual in the same household outbreak. On the basis of longitudinal isolates from individuals or households, we estimated an evolutionary rate of 0·5 SNPs per genome per year, consistent with a maximum of five SNPs between related isolates 3 years or less apart. Using a greater than five SNP threshold to assess 11 MIRU-VNTR-based community clusters, we found that none of 69 epidemiologically related pairs of MIRU-VNTR-matched cases plausibly related by transmission, two of 13 possibly related pairs, and 13 of 75 pairs with no known epidemiological relation were separated by more than five SNPs (p<0·0001). Seven MIRU-VNTR-matched pairs with no epidemiological relation had more than 30 SNPs, five of seven belonging to the same immigrant community cluster. WGS also showed that 62 of 75 MIRU-VNTR-matched pairs for which no epidemiological relation had been identified from contact tracing were highly likely to indicate transmission: in one substance misuse cluster, 38 individuals were linked by five or fewer SNPs without a single epidemiological link having been established previously. Further analysis suggested that microevolutionary divergence of lineages within outbreaks could signal possible super-spreaders, corroborated by clinical and epidemiological data in two clusters. InterpretationWGS can delineate tuberculosis outbreaks with greater resolution than has previously been possible. These findings offer public health teams the potential to limit outbreak investigations to patients who are likely to be linked by recent transmission, irrespective of whether it has been possible to identify epidemiological links, and to save resources where they are not, even in the context of matched MIRU-VNTR genotypes. Uniquely, WGS also provides information about the genetic structure of outbreak clusters, thereby providing the potential to direct public health resources towards individuals most likely to have infected the largest number of secondary cases. As a consequence, the Health Protection Agency is considering introduction of WGS technology for routine tuberculosis public health practice in England. FundingNIHR Oxford Biomedical Research Centre and the UKCRC Modernising Medical Microbiology Consortium (UKCRC Translational Infection Research Initiative supported by MRC, Biotechnology and Biological Sciences Research Council, and NIHR on behalf of the Department of Health [grant G0800778] and the Wellcome Trust [087646/Z/08/Z]).