scholarly journals MIP-MAP: High Throughput Mapping of Caenorhabditis elegans Temperature Sensitive Mutants via Molecular Inversion Probes

2017 ◽  
Author(s):  
CA Mok ◽  
V Au ◽  
OA Thompson ◽  
ML Edgley ◽  
L Gevirtzman ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Essential Genes ◽  
Model Organisms ◽  
Temperature Sensitive ◽  
Sequence Variant ◽  
Mutant Selection ◽  
Molecular Inversion Probes

AbstractTemperature sensitive (TS) alleles are important tools for the genetic and functional analysis of essential genes in many model organisms. While isolating TS alleles is not difficult, determining the TS-conferring mutation can be problematic. Even with whole-genome sequencing (WGS) data there is a paucity of predictive methods for identifying TS alleles from DNA sequence alone. We assembled 173 TS lethal mutants of Caenorhabditis elegans and used WGS to identify several hundred mutations per strain. We leveraged single molecule molecular inversion probes (MIPs) to sequence variant sites at high depth in the cross-progeny of TS mutants and a mapping strain with identified sequence variants but no apparent phenotypic differences from the reference N2 strain. By sampling for variants at ~1Mb intervals across the genome we genetically mapped mutant alleles at a resolution comparable to current standards in a process we call MIP-MAP. The MIP-MAP protocol, however, permits high-throughput sequencing of multiple TS mutation mapping libraries at less than 200K reads per library. Using MIP-MAP on a subset of TS mutants, via a competitive selection assay and standard recombinant mutant selection, we defined TS-associated intervals of 3Mb or less. Our results suggest this collection of strains contains a diverse library of TS alleles for genes involved in development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes. The MIPs protocol should allow high-throughput tracking of genetic variants in any mixed population.

scholarly journals MIP-MAP: High Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes

Genetics ◽  
2017 ◽  
pp. genetics.300179.2017 ◽  
Author(s):  
Calvin A. Mok ◽  
Vinci Au ◽  
Owen A. Thompson ◽  
Mark L. Edgley ◽  
Louis Gevirtzman ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Temperature Sensitive ◽  
Temperature Sensitive Mutants ◽  
Molecular Inversion Probes

scholarly journals Essential Genes Predicted in the Genome of Rubrivivax gelatinosus

2016 ◽  
Vol 198 (16) ◽  
pp. 2244-2250 ◽  
Author(s):  
Patrick D. Curtis
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Sugar Metabolism ◽  
Transposon Mutagenesis ◽  
Gas Production ◽  
Essential Genes ◽  
Complex Situation ◽  
Content Type ◽  
Physiological Characterization ◽  
Rubrivivax Gelatinosus

ABSTRACTRubrivivax gelatinosusis a betaproteobacterium with impressive metabolic diversity. It is capable of phototrophy, chemotrophy, two different mechanisms of sugar metabolism, fermentation, and H2gas production. To identify core essential genes,R. gelatinosuswas subjected to saturating transposon mutagenesis and high-throughput sequencing (TnSeq) analysis using nutrient-rich, aerobic conditions. Results revealed that virtually no primary metabolic genes are essential to the organism and that genomic redundancy only explains a portion of the nonessentiality, but some biosynthetic pathways are still essential under nutrient-rich conditions. Different essentialities of different portions of the Pho regulatory pathway suggest that overexpression of the regulon is toxic and hint at a larger connection between phosphate regulation and cellular health. Lastly, various essentialities of different tRNAs hint at a more complex situation than would be expected for such a core process. These results expand upon research regarding cross-organism gene essentiality and further enrich the study of purple nonsulfur bacteria.IMPORTANCEMicrobial genomic data are increasing at a tremendous rate, but physiological characterization of those data lags far behind. One mechanism of high-throughput physiological characterization is TnSeq, which uses high-volume transposon mutagenesis and high-throughput sequencing to identify all of the essential genes in a given organism's genome. Here TnSeq was used to identify essential genes in the metabolically versatile betaproteobacteriumRubrivivax gelatinosus. The results presented here add to the growing TnSeq field and also reveal important aspects ofR. gelatinosusphysiology, which are applicable to researchers working on metabolically flexible organisms.

scholarly journals Combined flow cytometry and high-throughput image analysis for the study of essential genes in Caenorhabditis elegans

BMC Biology ◽  
2018 ◽  
Vol 16 (1) ◽  
Author(s):  
Blanca Hernando-Rodríguez ◽  
Annmary Paul Erinjeri ◽  
María Jesús Rodríguez-Palero ◽  
Val Millar ◽  
Sara González-Hernández ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Image Analysis ◽  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Essential Genes ◽  
Combined Flow

scholarly journals A Caenorhabditis elegans RNA polymerase II gene, ama-1 IV, and nearby essential genes.

Genetics ◽  
1988 ◽  
Vol 118 (1) ◽  
pp. 61-74
Author(s):  
T M Rogalski ◽  
D L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gamma Ray ◽  
Temperature Shift ◽  
Developmental Rate ◽  
Egg Laying ◽  
Essential Genes ◽  
Sensitive Period ◽  
Temperature Sensitive

Abstract The amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans is encoded by the ama-1 gene, located approximately 0.05 map unit to the right of dpy-13 IV. Using the amanitin-resistant ama-1(m118) strain as a parent, we have isolated amanitin-sensitive mutants that carry recessive-lethal ama-1 alleles. Of the six ethyl methanesulfonate-induced mutants examined, two are arrested late in embryogenesis. One of these is a large deficiency, mDf9, but the second may be a novel point mutation. The four other mutants are hypomorphs, and presumably produce altered RNA polymerase II enzymes with some residual function. Two of these mutants develop into sterile adults at 20 degrees but are arrested as larvae at 25 degrees, and two others are fertile at 20 degrees and sterile at 25 degrees. Temperature-shift experiments performed with the adult sterile mutant, ama-1(m118m238ts), have revealed a temperature-sensitive period that begins late in gonadogenesis and is centered around the initiation of egg-laying. Postembryonic development at 25 degrees is slowed by 30%. By contrast, the amanitin-resistant allele of ama-1 has very little effect on developmental rate or fertility. We have identified 15 essential genes in an interval of 4.5 map units surrounding ama-1, as well as four gamma-ray-induced deficiencies and two duplications that include the ama-1 gene. The larger duplication, mDp1, may include the entire left arm of chromosome IV, and it recombines with the normal homologue at a low frequency. The smallest deficiency, mDf10, complements all but three identified genes: let-278, dpy-13 and ama-1, which define an interval of only 0.1 map unit. The terminal phenotype of mDf10 homozygotes is developmental arrest during the first larval stage, suggesting that there is sufficient maternal RNA polymerase II to complete embryonic development.

scholarly journals Population genomics of bank vole populations reveals associations between immune related genes and the epidemiology of Puumala hantavirus in Sweden

2017 ◽  
Author(s):  
Audrey Rohfritsch ◽  
Maxime Galan ◽  
Mathieu Gautier ◽  
Karim Gharbi ◽  
Gert Olsson ◽  
...  
Keyword(s):  
High Throughput ◽  
Bank Vole ◽  
High Throughput Sequencing ◽  
Population Genomics ◽  
Reservoir Host ◽  
Model Organisms ◽  
Myodes Glareolus ◽  
A Genome ◽  
Outlier Loci ◽  
Immune Related Genes

AbstractInfectious pathogens are major selective forces acting on individuals. The recent advent of high-throughput sequencing technologies now enables to investigate the genetic bases of resistance/susceptibility to infections in non-model organisms. From an evolutionary perspective, the analysis of the genetic diversity observed at these genes in natural populations provides insight into the mechanisms maintaining polymorphism and their epidemiological consequences. We explored these questions in the context of the interactions between Puumala hantavirus (PUUV) and its reservoir host, the bank vole Myodes glareolus. Despite the continuous spatial distribution of M. glareolus in Europe, PUUV distribution is strongly heterogeneous. Different defence strategies might have evolved in bank voles as a result of co-adaptation with PUUV, which may in turn reinforce spatial heterogeneity in PUUV distribution. We performed a genome scan study of six bank vole populations sampled along a North/South transect in Sweden, including PUUV endemic and non-endemic areas. We combined candidate gene analyses (Tlr4, Tlr7, Mx2 genes) and high throughput sequencing of RAD (Restriction-site Associated DNA) markers. We found evidence for outlier loci showing high levels of genetic differentiation. Ten outliers among the 52 that matched to mouse protein-coding genes corresponded to immune related genes and were detected using ecological associations with variations in PUUV prevalence. One third of the enriched pathways concerned immune processes, including platelet activation and TLR pathway. In the future, functional experimentations should enable to confirm the role of these these immune related genes with regard to the interactions between M. glareolus and PUUV.

scholarly journals Single Molecule Molecular Inversion Probes for High Throughput Germline Screenings in Dystonia

2019 ◽  
Vol 10 ◽  
Author(s):  
Michaela Pogoda ◽  
Franz-Joachim Hilke ◽  
Ebba Lohmann ◽  
Marc Sturm ◽  
Florian Lenz ◽  
...  
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Molecular Inversion Probes

scholarly journals IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Daniel F. Schorderet ◽  
Alexandra Iouranova ◽  
Tatiana Favez ◽  
Leila Tiab ◽  
Pascal Escher
Keyword(s):  
Retinitis Pigmentosa ◽  
Molecular Diagnosis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Variant ◽  
Sequence Variants ◽  
Sequencing Platform ◽  
Capture Assay ◽  
Retinal Dystrophies ◽  
Exon Capture

The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified inABCA4,CNGB1,GUCY2D,PROM1,PRPF8,PRPF31,PRPH2,RHO,RP2, andTULP1for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies.

scholarly journals Rapid isolation and characterization of microsatellites in the critically endangered Mountain Bongo (Tragelaphus eurycerus isaaci)

2019 ◽  
Author(s):  
Fraser John Combe ◽  
Evelyn Taylor cox ◽  
Graeme fox ◽  
Tommy Sandri ◽  
Bradley Cain ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Population Analysis ◽  
Illumina Miseq ◽  
Model Organisms ◽  
Conservation Genomics ◽  
Adaptive Genetic Variation ◽  
Genetics Research ◽  
Isolation And Characterization ◽  
Neutral Markers

High-throughput sequencing tools promise to revolutionize many aspects of genetics research, e.g. by allowing the identification of functional adaptive genetic variation. However, the expense and expertise required to apply these tools to basic conservation questions is a challenge for applications outside academia, resulting in a so-called “conservation genomics gap” (Shafer et al. 2015). The conservation genetics paradigm is that basic information about inbreeding and gene flow are often critical to inform conservation management of small populations (Ouborg et al. 2010). This information is often needed quickly and ideally should be accessible to workers without special expertise in genomics (DeSalle & Amato 2004). While the inferential power of high-throughput sequencing to interrogate the genome is profound, the cost for population analysis is higher (though decreasing) than for traditional neutral markers. Thus, the use of neutral markers is still relevant in conservation applications. However, this assumes that neutral markers have been discovered and characterized for a given species of conservation concern, which is often untrue for non-model organisms. Here, we use a fast, cost-efficient, high-throughput sequencing method (Illumina MiSeq) to rapidly identify and characterize microsatellites in the mountain bongo (Tragelaphus eurycerus isaaci, hereafter bongo), which has a clear and timely conservation imperative but lacks any described neutral markers.

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