scholarly journals Corrigendum and follow-up: Whole genome sequencing of multiple CRISPR-edited mouse lines suggests no excess mutations

2017 ◽  
Author(s):  
Kellie A. Schaefer ◽  
Benjamin W Darbro ◽  
Diana F. Colgan ◽  
Stephen H. Tsang ◽  
Alexander G. Bassuk ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Mouse Line ◽  
Genome Data ◽  
Parental Lines ◽  
Mouse Lines

Our previous publication suggested CRISPR-Cas9 editing at the zygotic stage might unexpectedly introduce a multitude of subtle but unintended mutations, an interpretation that not surprisingly raised numerous questions. The key issue is that since parental lines were not available, might the reported variants have been inherited? To expand upon the limited available whole genome data on whether CRISPR-edited mice show more genetic variation, whole-genome sequencing was performed on two other mouse lines that had undergone a CRISPR-editing procedure. Again, parents were not available for either the Capn5 nor Fblim1 CRISPR-edited mouse lines, so strain controls were examined. Additionally, we also include verification of variants detected in the initial mouse line. Taken together, these whole-genome-sequencing-level results support the idea that in specific cases, CRISPR-Cas9 editing can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

2021 ◽  
Author(s):  
◽  
Mariah Taylor ◽  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Animal Health ◽  
Functional Domains ◽  
Whole Genome ◽  
Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

A 10-year follow-up of a child with mild case of xeroderma pigmentosum complementation group D diagnosed by whole-genome sequencing

2016 ◽  
Vol 32 (4) ◽  
pp. 174-180 ◽  
Author(s):  
Ryusuke Ono ◽  
Taro Masaki ◽  
Franklin Mayca Pozo ◽  
Yuka Nakazawa ◽  
Sigrid M. A. Swagemakers ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Xeroderma Pigmentosum ◽  
Complementation Group ◽  
Whole Genome ◽  
Mild Case ◽  
Group D

scholarly journals Utility of Combining Whole Genome Sequencing with Traditional Investigational Methods To Solve Foodborne Outbreaks of Salmonella Infections Associated with Chicken: A New Tool for Tackling This Challenging Food Vehicle

2017 ◽  
Vol 80 (4) ◽  
pp. 654-660 ◽  
Author(s):  
Samuel J. Crowe ◽  
Alice Green ◽  
Kimberly Hernandez ◽  
Vi Peralta ◽  
Lyndsay Bottichio ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Food Samples ◽  
Clinical Isolates ◽  
Whole Genome ◽  
Salmonella Infections ◽  
Outbreak Investigations ◽  
Consumption Rates ◽  
Foodborne Outbreaks

ABSTRACT High consumption rates and a multitude of brands make multistate foodborne outbreaks of Salmonella infections associated with chicken challenging to investigate, but whole genome sequencing is a powerful tool that can be used to assist investigators. Whole genome sequencing of pathogens isolated from clinical, environmental, and food samples is increasingly being used in multistate foodborne outbreak investigations to determine with unprecedented resolution how closely related these isolates are to one another genetically. In 2014, federal and state health officials investigated an outbreak of 146 Salmonella Heidelberg infections in 24 states. A follow-up analysis was conducted after the conclusion of the investigation in which 27 clinical and 24 food isolates from the outbreak underwent whole genome sequencing. These isolates formed seven clades, the largest of which contained clinical isolates from a subcluster of case patients who attended a catered party. One isolate from a chicken processed by a large producer was closely related genetically (zero to three single-nucleotide polymorphism differences) to the clinical isolates from these subcluster case patients. Chicken from this large producer was also present in the kitchen of the caterer on the day before the event, thus providing additional evidence that the chicken from this producer was the outbreak source. This investigation highlights how whole genome sequencing can be used with epidemiologic and traceback evidence to identify chicken sources of foodborne outbreaks.

scholarly journals Analyses of X-linked and autosomal genetic variation in population-scale whole genome sequencing

2011 ◽  
Vol 43 (8) ◽  
pp. 741-743 ◽  
Author(s):  
Srikanth Gottipati ◽  
Leonardo Arbiza ◽  
Adam Siepel ◽  
Andrew G Clark ◽  
Alon Keinan
Keyword(s):  
Genetic Variation ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Population Scale

scholarly journals Whole-genome sequencing for an enhanced understanding of genetic variation among South Africans

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Ananyo Choudhury ◽  
Michèle Ramsay ◽  
Scott Hazelhurst ◽  
Shaun Aron ◽  
Soraya Bardien ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
South Africans

scholarly journals Identifying TCDD-resistance genes via murine and rat comparative genomics and transcriptomics

2019 ◽  
Author(s):  
Stephenie D. Prokopec ◽  
Aileen Lu ◽  
Sandy Che-Eun S. Lee ◽  
Cindy Q. Yao ◽  
Ren X. Sun ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Transgenic Mouse ◽  
Genome Sequencing ◽  
Mrna Abundance ◽  
Model Organisms ◽  
Whole Genome ◽  
Contributing Factors ◽  
Rat Models ◽  
Toxic Responses ◽  
Mouse Lines

AbstractThe aryl hydrocarbon receptor (AHR) mediates many of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, the AHR alone is insufficient to explain the widely different outcomes among organisms. Attempts to identify unknown factor(s) have been confounded by genetic variability of model organisms. Here, we evaluated three transgenic mouse lines, each expressing a different rat AHR isoform (rWT, DEL, and INS), as well as C57BL/6 and DBA/2 mice. We supplement these with whole-genome sequencing and transcriptomic analyses of the corresponding rat models: Long-Evans (L-E) and Han/Wistar (H/W) rats. These integrated multi-species genomic and transcriptomic data were used to identify genes associated with TCDD-response phenotypes.We identified several genes that show consistent transcriptional changes in both transgenic mice and rats. Hepatic Pxdc1 was significantly repressed by TCDD in C57BL/6, rWT mice, and in L-E rat. Three genes demonstrated different AHRE-1 (full) motif occurrences within their promoter regions: Cxxc5 had fewer occurrences in H/W, as compared with L-E; Sugp1 and Hgfac (in either L-E or H/W respectively). These genes also showed different patterns of mRNA abundance across strains.The AHR isoform explains much of the transcriptional variability: up to 50% of genes with altered mRNA abundance following TCDD exposure are associated with a single AHR isoform (30% and 10% unique to DEL and rWT respectively following 500 μg/kg TCDD). Genomic and transcriptomic evidence allowed identification of genes potentially involved in phenotypic outcomes: Pxdc1 had differential mRNA abundance by phenotype; Cxxc5 had altered AHR binding sites and differential mRNA abundance.Author SummaryEnvironmental contaminants such as dioxins cause many toxic responses, anything from chloracne (common in humans) to death. These toxic responses are mostly regulated by the Ahr, a ligand-activated transcription factor with roles in drug metabolism and immune responses, however other contributing factors remain unclear. Studies are complicated by the underlying genetic heterogeneity of model organisms. Our team evaluated a number of mouse and rat models, including two strains of mouse, two strains of rat and three transgenic mouse lines which differ only at the Ahr locus, that present widely different sensitivities to the most potent dioxin: 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD). We identified a number of changes to gene expression that were associated with different toxic responses. We then contrasted these findings with results from whole-genome sequencing of the H/W and L-E rats and found some key genes, such as Cxxc5 and Mafb, which might contribute to TCDD toxicity. These transcriptomic and genomic datasets will provide a valuable resource for future studies into the mechanisms of dioxin toxicities.

scholarly journals Mid-pass whole genome sequencing enables biomedical genetic studies of diverse populations

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Anne-Katrin Emde ◽  
Amanda Phipps-Green ◽  
Murray Cadzow ◽  
C. Scott Gallagher ◽  
Tanya J. Major ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Diverse Populations ◽  
Human Genetic Variation ◽  
Underrepresented Populations ◽  
Financial Barriers ◽  
Genetic Studies ◽  
Disease Relevance

Abstract Background Historically, geneticists have relied on genotyping arrays and imputation to study human genetic variation. However, an underrepresentation of diverse populations has resulted in arrays that poorly capture global genetic variation, and a lack of reference panels. This has contributed to deepening global health disparities. Whole genome sequencing (WGS) better captures genetic variation but remains prohibitively expensive. Thus, we explored WGS at “mid-pass” 1-7x coverage. Results Here, we developed and benchmarked methods for mid-pass sequencing. When applied to a population without an existing genomic reference panel, 4x mid-pass performed consistently well across ethnicities, with high recall (98%) and precision (97.5%). Conclusion Compared to array data imputed into 1000 Genomes, mid-pass performed better across all metrics and identified novel population-specific variants with potential disease relevance. We hope our work will reduce financial barriers for geneticists from underrepresented populations to characterize their genomes prior to biomedical genetic applications.

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