Coronavirus disease 2019 (COVID-19) outbreak on an in-patient medical unit associated with unrecognized exposures in common areas-Epidemiological and whole-genome sequencing investigation.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hospital outbreaks have been common and devastating during the coronavirus disease 2019 (COVID-19) pandemic. Understanding SARS-CoV-2 transmission in these environments is critical for preventing and managing outbreaks. DESIGN: Outbreak investigation through epidemiological mapping and whole-genome sequencing phylogeny. SETTING: Hospital in-patient medical unit outbreak in Toronto, Canada, from November 2020 to January 2021. PARTICIPANTS: The outbreak involved 8 patients and 10 staff and was associated with 3 patient deaths. RESULTS: Patients being cared for in geriatric chairs at the nursing station were at high risk for both acquiring and transmitting SARS-CoV-2 to other patients and staff. Furthermore, given the informal nature of these transmissions, they were not initially recognized, which led to further transmission and missing the opportunity for preventative COVID-19 therapies. CONCLUSIONS: During outbreak prevention and management, the risk of informal patient care settings, such as geriatric chairs, should be considered. During high-risk periods or during outbreaks, efforts should be made to care for patients in their rooms when possible.

Emergence of a mutation in the nucleocapsid gene of SARS-CoV-2 interferes with PCR detection in Canada.

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) was met with rapid development of robust molecular-based detection assays. Many SARS-CoV-2 molecular tests target multiple genetic regions of the virus to maximize detection and protect against diagnostic escape. Despite the relatively moderate mutational rate of SARS-CoV-2, numerous mutations with known negative impact on diagnostic assays have been identified. In early 2021, we identified four samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing revealed a single common mutation in the N gene C29200T. Spatiotemporal analysis showed that the mutation was found in at least six different Canadian provinces from May 2020 until May 2021. Phylogenetic analysis showed that this mutation arose multiple times in Canadian samples and is present in six different variants of interest and of concern. The Cepheid testing platform is commonly used in Canada including in remote regions. As such, the existence of N gene mutation dropouts required further investigation. While commercial SARS-CoV-2 molecular detection assays have contributed immensely to the response effort, many vendors are reluctant to make primer/probe sequences publicly available. Proprietary primer/probe sequences create diagnostic 'blind spots' for global SARS-CoV-2 sequence monitoring and limits the ability to detect and track the presence and prevalence of diagnostic escape mutations. We hope that our industry partners will seriously consider making primer/probe sequences available, so that diagnostic escape mutants can be identified promptly and responded to appropriately to maintain diagnostic accuracy.

Author Correction: Nanopore native RNA sequencing of a human poly(A) transcriptome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nanopore native RNA sequencing of a human poly(A) transcriptome.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.

Comparative toxicoproteogenomics of mouse and rat liver identifies TCDD-resistance genes.

The aryl hydrocarbon receptor (AHR) mediates many toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone does not explain the widely different outcomes among organisms. To identify the other factors involved, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS) providing widely different resistance to TCDD toxicity, as well as C57BL/6 and DBA/2 mice which exhibit a ~ tenfold divergence in TCDD sensitivity (exposures of 5-1000 µg/kg TCDD). We supplement these with whole-genome sequencing, together with transcriptomic and proteomic analyses of the corresponding rat models, Long-Evans (L-E) and Han/Wistar (H/W) rats (having a ~ 1000-fold difference in their TCDD sensitivities; 100 µg/kg TCDD), to identify genes associated with TCDD-response phenotypes. Overall, we identified up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (33.8%, 11.7%, 5.2% and 0.3% of 3076 genes altered unique to rWT, DEL, C57BL/6 and INS respectively following 1000 µg/kg TCDD). Hepatic Pxdc1 was significantly repressed in all three TCDD-sensitive animal models (C57BL/6 and rWT mice, and L-E rat) after TCDD exposure. Three genes, including Cxxc5, Sugp1 and Hgfac, demonstrated different AHRE-1 (full) motif occurrences within their promoter regions between rat strains, as well as different patterns of mRNA abundance. Several hepatic proteins showed parallel up- or downward alterations with their RNAs, with three genes (SNRK, IGTP and IMPA2) showing consistent, strain-dependent changes. These data show the value of integrating genomic, transcriptomic and proteomic evidence across multi-species models in toxicologic studies.

Sensitive tumour detection and classification using plasma cell-free DNA methylomes.

The use of liquid biopsies for cancer detection and management is rapidly gaining prominence1. Current methods for the detection of circulating tumour DNA involve sequencing somatic mutations using cell-free DNA, but the sensitivity of these methods may be low among patients with early-stage cancer given the limited number of recurrent mutations2-5. By contrast, large-scale epigenetic alterations-which are tissue- and cancer-type specific-are not similarly constrained6 and therefore potentially have greater ability to detect and classify cancers in patients with early-stage disease. Here we develop a sensitive, immunoprecipitation-based protocol to analyse the methylome of small quantities of circulating cell-free DNA, and demonstrate the ability to detect large-scale DNA methylation changes that are enriched for tumour-specific patterns. We also demonstrate robust performance in cancer detection and classification across an extensive collection of plasma samples from several tumour types. This work sets the stage to establish biomarkers for the minimally invasive detection, interception and classification of early-stage cancers based on plasma cell-free DNA methylation patterns.

Prediction of acute myeloid leukaemia risk in healthy individuals.

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure1. The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion2,3. However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH)4-8. Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention.

Minimally Invasive Real-Time Detection of Actionable Mutations in Patients With Metastatic Solid Tumors Using Fine-Needle and Liquid Biopsies.

PURPOSE: Fine-needle biopsy (FNB) and liquid biopsy are minimally invasive methods of tumor sampling that provide feasible means to assess tumor genotypes in real time. However, more data are needed to establish the strength of these methods by benchmarking against the current gold standard methods, core-needle biopsy (CNB) or surgical excision of the tumor. PATIENTS AND METHODS: Eligible patients with advanced solid tumors were prospectively recruited. We performed mutation profiling using matched tumor DNA obtained by CNB, FNB and liquid biopsy, and matrix-assisted laser desorption/ionization time-of-flight custom mass-spectrometry or targeted next-generation DNA sequencing. The actionability of detected mutations was determined using the OncoKB Web tool. Agreement between mutations detected in CNBs, FNBs, and circulating tumor DNA (ctDNA) was examined. RESULTS: Forty-one patients underwent tumor biopsy. Thirty CNBs (73%) and 34 FNBs (83%) had sufficient tumor and DNA for mutation profiling. Median DNA yield from CNB and FNB were 775 ng (interquartile range, 240 to 347 4ng) and 649 ng (interquartile range, 180 to1350 ng), respectively. Of 29 CNB/FNB pairs available for comparison, actionable mutation results were concordant in 28 (96%). Six of nine actionable mutations (67%) that were found by CNB, FNB, or both were detectable in ctDNA. Two additional actionable mutations were found exclusively in ctDNA. CONCLUSION: Optimally processed FNB and liquid biopsy can be used routinely for tumor mutation profiling to identify actionable mutations.

Fine mapping of chromosome 5p15.33 based on a targeted deep sequencing and high density genotyping identifies novel lung cancer susceptibility loci.

Chromosome 5p15.33 has been identified as a lung cancer susceptibility locus, however the underlying causal mechanisms were not fully elucidated. Previous fine-mapping studies of this locus have relied on imputation or investigated a small number of known, common variants. This study represents a significant advance over previous research by investigating a large number of novel, rare variants, as well as their underlying mechanisms through telomere length. Variants for this fine-mapping study were identified through a targeted deep sequencing (average depth of coverage greater than 4000×) of 576 individuals. Subsequently, 4652 SNPs, including 1108 novel SNPs, were genotyped in 5164 cases and 5716 controls of European ancestry. After adjusting for known risk loci, rs2736100 and rs401681, we identified a new, independent lung cancer susceptibility variant in LPCAT1: rs139852726 (OR = 0.46, P = 4.73×10(-9)), and three new adenocarcinoma risk variants in TERT: rs61748181 (OR = 0.53, P = 2.64×10(-6)), rs112290073 (OR = 1.85, P = 1.27×10(-5)), rs138895564 (OR = 2.16, P = 2.06×10(-5); among young cases, OR = 3.77, P = 8.41×10(-4)). In addition, we found that rs139852726 (P = 1.44×10(-3)) was associated with telomere length in a sample of 922 healthy individuals. The gene-based SKAT-O analysis implicated TERT as the most relevant gene in the 5p15.33 region for adenocarcinoma (P = 7.84×10(-7)) and lung cancer (P = 2.37×10(-5)) risk. In this largest fine-mapping study to investigate a large number of rare and novel variants within 5p15.33, we identified novel lung and adenocarcinoma susceptibility loci with large effects and provided support for the role of telomere length as the potential underlying mechanism.

A two-dimensional pooling strategy for rare variant detection on next-generation sequencing platforms.

We describe a method for pooling and sequencing DNA from a large number of individual samples while preserving information regarding sample identity. DNA from 576 individuals was arranged into four 12 row by 12 column matrices and then pooled by row and by column resulting in 96 total pools with 12 individuals in each pool. Pooling of DNA was carried out in a two-dimensional fashion, such that DNA from each individual is present in exactly one row pool and exactly one column pool. By considering the variants observed in the rows and columns of a matrix we are able to trace rare variants back to the specific individuals that carry them. The pooled DNA samples were enriched over a 250 kb region previously identified by GWAS to significantly predispose individuals to lung cancer. All 96 pools (12 row and 12 column pools from 4 matrices) were barcoded and sequenced on an Illumina HiSeq 2000 instrument with an average depth of coverage greater than 4,000×. Verification based on Ion PGM sequencing confirmed the presence of 91.4% of confidently classified SNVs assayed. In this way, each individual sample is sequenced in multiple pools providing more accurate variant calling than a single pool or a multiplexed approach. This provides a powerful method for rare variant detection in regions of interest at a reduced cost to the researcher.

Monoubiquitylation of H2A.Z distinguishes its association with euchromatin or facultative heterochromatin.

H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z- or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.