We thank Jose Salazar, Julia Wang, and Dr. Karen Schulze for critical reading of the manuscript. We acknowledge Drs. Ning Liu and Xi Luo for the functional characterization of the TBX2 variants discussed here. Undiagnosed Diseases Network Model Organisms Screening Center was supported through the National Institutes of Health (NIH) Common Fund (U54 NS093793). H. T. C. was further supported by the NIH[CNCDP-K12 and NINDS (1K12 NS098482)], American Academy of Neurology (Neuroscience Research grant), Burroughs Wellcome Fund (Career Award for Medical Scientists), Child Neurology Society and Child Neurology Foundation (PERF Elterman grant), and the NIH Director&#8217;s Early Independence Award (DP5 OD026426). M. F. W. was further supported by Simons Foundation (SFARI Award: 368479). S. Y. was further supported by the NIH (R01 DC014932), the Simons Foundation (SFARI Award: 368479), the Alzheimer&#8217;s Association (New Investigator Research Grant: 15-364099), Naman Family Fund for Basic Research, and Caroline Wiess Law Fund for Research in Molecular Medicine. Confocal microscopy at BCM is supported in part by NIH Grant U54HD083092 to the Intellectual and Developmental Disabilities Research Center (IDDRC) Neurovisualization Core.

The authors have nothing to disclose.

Experimental studies using Drosophila melanogaster provide a robust assay system to assess the consequences of disease-associated human variants. This is due to the large body of knowledge and diverse genetic tools that have been generated by many researchers in the fly field over the past century89. Just like any other experimental system, however, it is important to acknowledge the caveats and limitations that exist.
Caveats Associated with Data Minin...

Patients with rare diseases often undergo an arduous journey referred to as the &#34;diagnostic odyssey&#34;&#160;to obtain an accurate diagnosis1. Most rare diseases are thought to have a strong genetic origin, making genetic/genomic analyses critical elements of the clinical workup. In addition to candidate gene panel sequencing and copy number variation analysis based on chromosomal microarrays, whole-exome (WES) and whole-genome sequencing (WGS) technologies have become increasingly valuable tools over the past decade2,3. Currently, the diagnostic rate for identifying a known pathogenic variant in WES and WGS is ~25% (higher in pediatric cases)4,5. For most cases that remain undiagnosed after clinical WES/WGS, a common issue is that there are many candidate genes and variants. Next-generation sequencing often identifies novel or ultra-rare variants in many genes, and interpreting whether these variants contribute to disease phenotypes is challenging. For example, although most nonsense or frameshift mutations in genes are thought to be loss-of-function (LOF) alleles due to nonsense-mediated decay of the encoded transcript, truncating mutations found in the last exons escape this process and may function as benign or gain-of-function (GOF) alleles6.
Moreover, predicting the effects of a missense allele is a daunting task, since it can result in a number of different genetic scenarios as first described by Herman Muller in the 1930s (i.e., amorph, hypomorph, hypermorph, antimorph, neomorph, or isomorph)7. Numerous in silico programs and methodologies have been developed to predict the pathogenicity of missense variants based on evolutionary conservation, type of amino acid change, position within a functional domain, allele frequency in the general population, and other parameters8. However, these programs are not a comprehensive solution to solving the complicated problem of variant interpretation. Interestingly, a recent study demonstrated that five broadly used variant pathogenicity prediction algorithms (Polyphen9, SIFT10, CADD11, PROVEAN12, Mutation Taster) agree on pathogenicity ~80% of the time8. Notably, even when all algorithms agree, they return an incorrect prediction of pathogenicity up to 11% of the time. This not only leads to flawed clinical interpretation but also may dissuade researchers from following up on new variants by falsely listing them as benign. One way to complement the current limitation of in silico modeling is to provide experimental data that demonstrates the effect of variant function in vitro, ex vivo (e.g., cultured cells, organoids), or in vivo.
In vivo functional studies of rare disease associated variants in MO have unique strengths13 and have been adopted by many rare disease research initiatives around the world, including the Undiagnosed Diseases Network (UDN) in the United States and Rare Diseases Models &#38; Mechanisms (RDMM) Networks in Canada, Japan, Europe, and Australia14. In addition to these coordinated efforts to integrate MO researchers into the workflow of rare disease diagnosis and mechanistic studies at a national scale, a number of individual collaborative studies between clinical and MO researchers have led to the discovery and characterization of many new human disease-causing genes and variants82,83,84.
In the UDN, a centralized Model Organisms Screening Center (MOSC) receives submissions of candidate genes and variants with a description of the patient&#8217;s condition and assesses whether the variant is likely to be pathogenic using informatics tools and in vivo experiments. In Phase I (2015-2018) of the UDN, the MOSC comprised of a Drosophila Core [Baylor College of Medicine (BCM)] and Zebrafish Core (University of Oregon) that worked collaboratively to assess cases. Using informatics analysis and a number of different experimental strategies in Drosophila and zebrafish, the MOSC has so far contributed to the diagnosis of 132 patients, identification of 31 new syndromes55, discovery of several new human disease genes (e.g., EBF315, ATP5F1D16, TBX217, IRF2BPL18, COG419, WDR3720) and phenotypic expansion of known disease genes (e.g., CACNA1A21, ACOX122).
In addition to projects within the UDN, MOSC Drosophila Core researchers have contributed to new disease gene discoveries in collaboration with the Centers for Mendelian Genomics and other initiatives (e.g., ANKLE223, TM2D324, NRD125, OGDHL25, ATAD3A26, ARIH127, MARK328, DNMBP29) using the same set of informatics and genetic strategies developed for the UDN. Given the significance of MO studies on rare disease diagnosis, the MOSC was expanded to include a C. elegans Core and second Zebrafish core (both at Washington University at St. Louis) for Phase II (2018-2022) of the UDN.
This manuscript describes an in vivo functional study protocol that is actively used in the UDN MOSC Drosophila Core to determine if missense variants have functional consequences on the protein of interest using transgenic flies that express human proteins. The goal of this protocol is to help MO researchers work collaboratively with clinical research groups to provide experimental evidence that a candidate variant in a gene of interest has functional consequences, thus facilitating clinical diagnosis. This protocol is most useful in a scenario in which a Drosophila researcher is approached by a clinical investigator who has a rare disease patient with a specific candidate variant in a gene of interest.
This protocol can be broken down into three elements: (1) gathering information to assess the likelihood of the variant of interest being responsible for the patient phenotype and the feasibility of a functional study in Drosophila, (2) gathering existing genetic tools and establishing new ones, and (3) performing functional studies in vivo. The third element can further be subdivided into two sub-elements based on how the function of a variant of interest can be assessed (rescue experiment or overexpression-based strategies). It is important to note that this protocol can be adapted and optimized to many scenarios outside of rare monogenic disease research (e.g., common diseases, gene-environment interactions, and pharmacological/genetic screens to identify therapeutic targets). The ability to determine the functionality and pathogenicity of variants will not only benefit the patient of interest by providing accurate molecular diagnosis but will also have broader impacts on both translational and basic scientific research.

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			<td height="21" style="height:22px;width:705px;">Drosophila Stocks for UAS-human cDNA transgenesis</td>
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			<td height="54" style="height:54px;width:325px;">Injection strains for transgenesis (D. melanogaster)</td>
			<td style="width:189px;">BDSC</td>
			<td style="width:191px;">#24871</td>
			<td style="width:385px;">Specific Reagent: VK33 (3rd chromosome) Injection line</td>
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			<td height="54" style="height:54px;width:325px;">Injection strains for transgenesis (D. melanogaster)</td>
			<td height="25" style="height:25px;width:189px;">BDSC</td>
			<td style="width:191px;">#24872</td>
			<td style="width:385px;">Specific Reagent: VK37 (2nd chromosome) Injection line</td>
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			<td height="25" style="height:25px;width:705px;">Plasmid DNA</td>
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			<td height="25" style="height:25px;width:325px;">Cloning vector</td>
			<td style="width:189px;">Thermo Fisher</td>
			<td style="width:191px;">#12536-017</td>
			<td style="width:385px;">Specific Reagent: pDONR221</td>
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			<td height="25" style="height:25px;width:325px;">Drosophila transgenesis vector</td>
			<td style="width:380px;">Gift from Drs. Johannes Bischof and Konrad Basler (Bischof et al., 2013 PNAS)</td>
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			<td style="width:385px;">Specific Reagent: pGW-HA.attB</td>
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			<td height="25" style="height:25px;width:705px;">Molecular biology kits and reagents</td>
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			<td style="width:189px;">Sigma-Aldrich</td>
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			<td style="width:385px;">Specific Reagent: Agarose (molecular biology grade)</td>
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			<td height="25" style="height:25px;width:325px;">Chemically Competent Cells (E. coli)</td>
			<td style="width:189px;">Thermo Fisher</td>
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			<td height="25" style="height:25px;width:325px;">DNA Gel Extraction kit</td>
			<td style="width:189px;">Thermo Fisher</td>
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			<td style="width:385px;">Specific Reagent: PureLink Gel Extraction Kit</td>
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			<td height="25" style="height:25px;width:325px;">DNA Isolation and purification kit</td>
			<td style="width:189px;">Qiagen</td>
			<td style="width:191px;">#27104</td>
			<td style="width:385px;">Specific Reagent: QIAprep Spin Miniprep Kit</td>
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			<td height="25" style="height:25px;width:325px;">High Fidelity Polymerase</td>
			<td style="width:189px;">NEB</td>
			<td style="width:191px;">#M0491</td>
			<td style="width:385px;">Specific Reagent: Q5 Polymerase kit</td>
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			<td height="75" style="height:75px;width:325px;">Recombinase mediated cloning system</td>
			<td style="width:189px;">Thermo Fisher</td>
			<td style="width:191px;">#11789020</td>
			<td style="width:385px;">Specific Reagent: Gateway BP Clonase kit</td>
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			<td height="75" style="height:75px;width:325px;">Recombinase mediated cloning system</td>
			<td height="50" style="height:50px;width:189px;">Thermo Fisher</td>
			<td style="width:191px;">#11791100</td>
			<td style="width:385px;">Specific Reagent: Gateway LR Clonase II Enzyme kit</td>
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			<td height="50" style="height:50px;width:325px;">Site Directed Mutagenesis kit</td>
			<td style="width:189px;">Agilent</td>
			<td style="width:191px;">#200523</td>
			<td style="width:385px;">Specific Reagent: Quick Change II Mutagenesis kit</td>
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			<td height="25" style="height:25px;width:705px;">Electroretinogram Rig related equipment</td>
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			<td height="50" style="height:50px;width:325px;">ERG Analysis</td>
			<td style="width:189px;">Molecular Devices</td>
			<td style="width:191px;">N/A</td>
			<td style="width:385px;">Specific Reagent: Axon pCLAMP 10 Data Software Package</td>
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			<td height="50" style="height:50px;width:325px;">ERG Data Collection</td>
			<td style="width:189px;">LabX</td>
			<td style="width:191px;">#R150358</td>
			<td style="width:385px;">Specific Reagent: ISO-DAM Isolated Biologic Amplifier</td>
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			<td height="25" style="height:25px;width:325px;">ERG Stimulator</td>
			<td style="width:189px;">Astro-Med</td>
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			<td style="width:385px;">Specific Reagent: Square Pulse Stimulator</td>
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</table>

in vivo functional study of disease-associated rare human variants using drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

Functional Study of de novo Missense Variant in EBF3 Linked to Neurodevelopmental Phenotypes 
In a 7 year-old male with neurodevelopmental phenotypes including hypotonia, ataxia, global developmental delay, and expressive speech disorder, physicians and human geneticists at the National Institutes of Health Undiagnosed Diseases Project (UDP) identified a de novo missense variant (p.R163Q) in EBF3 (Early B-Cell Factor 3)15, a gene that encodes a COE ...

Watch this Scientific Journal Video about In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila at JoVE.com

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

in-vivo-functional-study-disease-associated-rare-human-variants-using

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JoVE Journal

Genetics

13.5K Views.  Baylor College of Medicine. The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Video: In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...