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

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.

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Utility of whole-genome sequencing during an investigation of multiple foodborne outbreaks of Shigella sonnei

Epidemiology and Infection ◽

10.1017/s0950268821000509 ◽

2021 ◽

Vol 149 ◽

Author(s):

Amy F. W. Mikhail ◽

Monique Pereboom ◽

Lara Utsi ◽

Jeremy Hawker ◽

Jonathan Lighthill ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Middle Eastern ◽

Epidemiological Data ◽

Multidrug Resistant ◽

Food Samples ◽

Shigella Sonnei ◽

Whole Genome ◽

Contributing Factors ◽

Outbreak Strain

Abstract In April 2018, Public Health England was notified of cases of Shigella sonnei who had eaten food from three different catering outlets in England. The outbreaks were initially investigated as separate events, but whole-genome sequencing (WGS) showed they were caused by the same strain. The investigation included analyses of epidemiological data, the food chain and microbiological examination of food samples. WGS was used to determine the phylogenetic relatedness and antimicrobial resistance profile of the outbreak strain. Ultimately, 33 cases were linked to this outbreak; the majority had eaten food from seven outlets specialising in Indian or Middle Eastern cuisine. Five outlets were linked to two or more cases, all of which used fresh coriander although a shared supplier was not identified. An investigation at one of the venues recorded that 86% of cases reported eating dishes with coriander as an ingredient or garnish. Four cases were admitted to hospital and one had evidence of treatment failure with ciprofloxacin. Phylogenetic analysis showed that the outbreak strain was part of a wider multidrug-resistant clade associated with travel to Pakistan. Poor hygiene practices during cultivation, distribution or preparation of fresh produce are likely contributing factors.

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Challenges and Approaches to Genotyping Repetitive DNA

G3 Genes|Genome|Genetics ◽

10.1534/g3.119.400771 ◽

2019 ◽

Vol 10 (1) ◽

pp. 417-430 ◽

Cited By ~ 2

Author(s):

Elizabeth A. Morton ◽

Ashley N. Hall ◽

Elizabeth Kwan ◽

Calvin Mok ◽

Konstantin Queitsch ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Repetitive Dna ◽

Complex Traits ◽

Copy Number ◽

Model Organisms ◽

Whole Genome ◽

Copy Number Estimation ◽

Rdna Copy ◽

Rdna Copy Number

Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

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Whole genome sequencing identifies a novel factor required for secretory granule maturation in Tetrahymena thermophila

10.1101/042085 ◽

2016 ◽

Author(s):

Cassandra Kontur ◽

Santosh Kumar ◽

Xun Lan ◽

Jonathan K Pritchard ◽

Aaron P Turkewitz

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Tetrahymena Thermophila ◽

Secretory Granules ◽

Protein A ◽

Model Organisms ◽

Whole Genome ◽

Lysosomal Sorting ◽

Granule Maturation ◽

Multiple Strains

Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded into a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies in part on ancestral lysosomal sorting machinery but is also likely to involve novel factors. In prior work, multiple strains with defect in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation, a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wildtype copy of MMA1, and disruption of MMA1 in an otherwise wildtype strain generated a phenocopy of UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation.

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Mapping challenging mutations by whole-genome sequencing

10.1101/036046 ◽

2016 ◽

Author(s):

Harold E. Smith ◽

Amy S. Fabritius ◽

Aimee Jaramillo-Lambert ◽

Andy Golden

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Single Gene ◽

Global Scale ◽

Model Organisms ◽

Whole Genome ◽

Genetic Screens ◽

Alternative Approach ◽

Mutation Identification

ABSTRACTWhole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homozygous variants). However, such approaches are not entirely suitable for the characterization of a variety of more challenging mutations, such as dominant and semi-dominant alleles or multigenic traits. Therefore, we have developed strategies for the identification of those classes of mutations, using polymorphism mapping in Caenorhabditis elegans as our model for validation. We also report an alternative approach for mutation identification from traditional recombinant crosses, and a solution to the technical challenge of sequencing sterile or terminally arrested strains where population size is limiting. The methods described herein extend the applicability of whole-genome sequencing to a broader spectrum of mutations, including classes that are difficult to map by traditional means.

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

10.1101/154450 ◽

2017 ◽

Cited By ~ 4

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.

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Genomic Sequencing To Identify Potential Causative Mutation(s) of Neurospora crassa col-4

Microbiology Resource Announcements ◽

10.1128/mra.01009-19 ◽

2020 ◽

Vol 9 (2) ◽

Author(s):

Thomas A. Randall

Keyword(s):

Neurospora Crassa ◽

Whole Genome Sequencing ◽

Genetic Markers ◽

Genome Sequencing ◽

Model Organisms ◽

Causative Mutation ◽

Genomic Sequencing ◽

Whole Genome ◽

Content Type

In many cases, genes for commonly used genetic markers in model organisms have not been identified; therefore, it is of interest to identify the causative genes. Whole-genome sequencing was used to identify potential causative mutations for a col-4 allele of Neurospora crassa.

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ViR: a tool to solve intrasample variability in the prediction of viral integration sites using whole genome sequencing data

BMC Bioinformatics ◽

10.1186/s12859-021-03980-5 ◽

2021 ◽

Vol 22 (1) ◽

Cited By ~ 1

Author(s):

Elisa Pischedda ◽

Cristina Crava ◽

Martina Carlassara ◽

Susanna Zucca ◽

Leila Gasmi ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Model Organisms ◽

Whole Genome Sequencing Data ◽

Whole Genome ◽

Sequencing Data ◽

Regulation Of Expression ◽

Transfer Event ◽

A Genome ◽

Integration Sites

Abstract Background Several bioinformatics pipelines have been developed to detect sequences from viruses that integrate into the human genome because of the health relevance of these integrations, such as in the persistence of viral infection and/or in generating genotoxic effects, often progressing into cancer. Recent genomics and metagenomics analyses have shown that viruses also integrate into the genome of non-model organisms (i.e., arthropods, fish, plants, vertebrates). However, rarely studies of endogenous viral elements (EVEs) in non-model organisms have gone beyond their characterization from reference genome assemblies. In non-model organisms, we lack a thorough understanding of the widespread occurrence of EVEs and their biological relevance, apart from sporadic cases which nevertheless point to significant roles of EVEs in immunity and regulation of expression. The concomitance of repetitive DNA, duplications and/or assembly fragmentations in a genome sequence and intrasample variability in whole-genome sequencing (WGS) data could determine misalignments when mapping data to a genome assembly. This phenomenon hinders our ability to properly identify integration sites. Results To fill this gap, we developed ViR, a pipeline which solves the dispersion of reads due to intrasample variability in sequencing data from both single and pooled DNA samples thus ameliorating the detection of integration sites. We tested ViR to work with both in silico and real sequencing data from a non-model organism, the arboviral vector Aedes albopictus. Potential viral integrations predicted by ViR were molecularly validated supporting the accuracy of ViR results. Conclusion ViR will open new venues to explore the biology of EVEs, especially in non-model organisms. Importantly, while we generated ViR with the identification of EVEs in mind, its application can be extended to detect any lateral transfer event providing an ad-hoc sequence to interrogate.

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Deciphering complex genome rearrangements in C. elegans using short-read whole genome sequencing

Scientific Reports ◽

10.1038/s41598-021-97764-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tatiana Maroilley ◽

Xiao Li ◽

Matthew Oldach ◽

Francesca Jean ◽

Susan J. Stasiuk ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Chromosomal Rearrangements ◽

Large Deletion ◽

Genomic Rearrangements ◽

Model Organisms ◽

Whole Genome ◽

Short Read ◽

C Elegans ◽

Long Read

AbstractGenomic rearrangements cause congenital disorders, cancer, and complex diseases in human. Yet, they are still understudied in rare diseases because their detection is challenging, despite the advent of whole genome sequencing (WGS) technologies. Short-read (srWGS) and long-read WGS approaches are regularly compared, and the latter is commonly recommended in studies focusing on genomic rearrangements. However, srWGS is currently the most economical, accurate, and widely supported technology. In Caenorhabditis elegans (C. elegans), such variants, induced by various mutagenesis processes, have been used for decades to balance large genomic regions by preventing chromosomal crossover events and allowing the maintenance of lethal mutations. Interestingly, those chromosomal rearrangements have rarely been characterized on a molecular level. To evaluate the ability of srWGS to detect various types of complex genomic rearrangements, we sequenced three balancer strains using short-read Illumina technology. As we experimentally validated the breakpoints uncovered by srWGS, we showed that, by combining several types of analyses, srWGS enables the detection of a reciprocal translocation (eT1), a free duplication (sDp3), a large deletion (sC4), and chromoanagenesis events. Thus, applying srWGS to decipher real complex genomic rearrangements in model organisms may help designing efficient bioinformatics pipelines with systematic detection of complex rearrangements in human genomes.

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First de-novo transcriptome assembly of a South American frog, Oreobates cruralis, enables population genomic studies of Neotropical amphibians

10.7287/peerj.preprints.2980v1 ◽

2017 ◽

Author(s):

Santiago Montero-Mendieta ◽

Manfred Grabherr ◽

Henrik Lantz ◽

Ignacio De la Riva ◽

Jennifer A Leonard ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

De Novo ◽

Transcriptome Assembly ◽

Cost Effective ◽

Model Organisms ◽

South American ◽

Whole Genome ◽

Rna Seq ◽

De Novo Transcriptome

Whole genome sequencing is opening the door to novel insights into the population structure and evolutionary history of poorly known species. In organisms with large genomes, which includes most amphibians, whole-genome sequencing is excessively challenging and transcriptome sequencing (RNA-seq) represents a cost-effective tool to explore genome-wide variability. Non-model organisms do not usually have a reference genome to facilitate assembly and the transcriptome sequence must be assembled de-novo. We used RNA-seq to obtain the transcriptome profile for Oreobates cruralis, a poorly known South American direct-developing frog. In total, 550,871 transcripts were assembled, corresponding to 422,999 putative genes. Of those, we identified 23,500, 37,349, 38,120 and 45,885 genes present in the Pfam, EggNOG, KEGG and GO databases, respectively. Interestingly, our results suggested that genes related to immune system and defense mechanisms are abundant in the transcriptome of O. cruralis. We also present a workflow to assist with pre-processing, assembling, evaluating and functionally annotating a de-novo transcriptome from RNA-seq data of non-model organisms. Our workflow guides the inexperienced user in an intuitive way through all the necessary steps to build de-novo transcriptome assemblies using readily available software and is freely available at: https://github.com/biomendi/PRACTICAL-GUIDE-TO-BUILD-DE-NOVO-TRANSCRIPTOME-ASSEMBLIES-FOR-NON-MODEL-ORGANISMS/wiki

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