Name: navigating marrvel, a web-based tool that integrates human genomics and model organism genetics information
Uploaded: 2019-08-15T13:00:00
Description: Watch this Scientific Journal Video about Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information at JoVE.com

We thank Drs. Rami Al-Ouran, Seon-Young Kim, Yanhui (Claire) Hu, Ying-Wooi Wan, Naveen Manoharan, Sasidhar Pasupuleti, Aram Comjean, Dongxue Mao, Michael Wangler, Hsiao-Tuan Chao, Stephanie Mohr, and Norbert Perrimon for their support in the development and maintenance of MARRVEL. We are grateful to Samantha L. Deal and J. Michael Harnish for their input on this manuscript. &#160;
The initial development of MARRVEL was supported in part by the Undiagnosed Diseases Network Model Organisms Screening Center through the NIH Commonfund (U54NS093793) and&#160;through the NIH Office of Research Infrastructure Programs (ORIP)&#160;(R24OD022005).&#160;JW is supported by the NIH Eunice Kennedy Shriver National Institute of Child Health &#38; Human Development (F30HD094503) and The Robert and Janice McNair Foundation McNair MD/PhD Student Scholar Program at BCM. HJB is further supported by the NIH National Institute of General Medical Sciences (R01GM067858) and is an Investigator of the Howard Hughes Medical Institute. ZL is supported by the NIH National Institute of General Medical Science (R01GM120033), National Institute of Aging (R01AG057339), and the Huffington Foundation. SY received additional support from the NIH National Institute on Deafness and other Communication Disorders (R01DC014932), 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.&#160;

1. How to begin a search
<ol>
	<li>For the human gene and variant-based search, go to steps 1.1.1.-1.1.2. For human gene-based search (no variant input), go to step 1.2. For model organism gene-based search, refer to steps 1.3.1.-1.3.2.
	<ol>
		<li>Go to the home page of MARRVEL4 at http://marrvel.org/. Start by entering a human gene symbol. Ensure that the candidate gene names are listed below the input box with each character entry. If the search comes back negative, make sure the gene symbol ...

<ol>
	<li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+whole-exome+sequencing+for+the+diagnosis+of+mendelian+disorders.">Clinical whole-exome sequencing for the diagnosis of mendelian disorders.</a> New England Journal of Medicine. 369 (16), 1502-1511 (2013).</li><li>Richards, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standards+and+guidelines+for+the+interpretation+of+sequence+variants:+a+joint+consensus+recommendation+of+the+American+College+of+Medical+Genetics+and+Genomics+and+the+Association+for+Molecular+Pathology.">Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.</a> Genetics in Medicine. 17 (5), 405-424 (2015).</li><li>MacArthur, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+investigating+causality+of+sequence+variants+in+human+disease.">Guidelines for investigating causality of sequence variants in human disease.</a> Nature. 508 (7497), 469-476 (2014).</li><li>Wang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MARRVEL:+Integration+of+Human+and+Model+Organism+Genetic+Resources+to+Facilitate+Functional+Annotation+of+the+Human+Genome.">MARRVEL: Integration of Human and Model Organism Genetic Resources to Facilitate Functional Annotation of the Human Genome.</a> American Journal of Human Genetics. 100 (6), 843-853 (2017).</li><li>Povey, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HUGO+Gene+Nomenclature+Committee+(HGNC).">The HUGO Gene Nomenclature Committee (HGNC).</a> Human Genetics. 109 (6), 678-680 (2001).</li><li>Lek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein-coding+genetic+variation+in+60,706+humans.">Analysis of protein-coding genetic variation in 60,706 humans.</a> Nature. 536 (7616), 285-291 (2016).</li><li>Wildeman, M., van Ophuizen, E., den Dunnen, J. T., Taschner, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+sequence+variant+descriptions+in+mutation+databases+and+literature+using+the+Mutalyzer+sequence+variation+nomenclature+checker.">Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker.</a> Human Mutation. 29 (1), 6-13 (2008).</li><li>Zhou, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TransVar:+a+multilevel+variant+annotator+for+precision+genomics.">TransVar: a multilevel variant annotator for precision genomics.</a> Nature Methods. 12 (11), 1002-1003 (2015).</li><li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrative+approach+to+ortholog+prediction+for+disease-focused+and+other+functional+studies.">An integrative approach to ortholog prediction for disease-focused and other functional studies.</a> BMC Bioinformatics. 12, 357 (2011).</li><li>Amberger, J. S., Hamosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+Online+Mendelian+Inheritance+in+Man+(OMIM):+A+Knowledgebase+of+Human+Genes+and+Genetic+Phenotypes.">Searching Online Mendelian Inheritance in Man (OMIM): A Knowledgebase of Human Genes and Genetic Phenotypes.</a> Current Protocols in Bioinformatics. 58, 1 (2017).</li><li>Amberger, J. S., Bocchini, C. A., Scott, A. F., Hamosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OMIM.org:+leveraging+knowledge+across+phenotype-gene+relationships.">OMIM.org: leveraging knowledge across phenotype-gene relationships.</a> Nucleic Acids Research. 47, 1038-1043 (2019).</li><li>Liu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+variants+in+TBX2+are+associated+with+a+syndromic+cardiovascular+and+skeletal+developmental+disorder.">Functional variants in TBX2 are associated with a syndromic cardiovascular and skeletal developmental disorder.</a> Human Molecular Genetics. 27 (14), 2454-2465 (2018).</li><li>Ropers, H. H., Wienker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Penetrance+of+pathogenic+mutations+in+haploinsufficient+genes+for+intellectual+disability+and+related+disorders.">Penetrance of pathogenic mutations in haploinsufficient genes for intellectual disability and related disorders.</a> European Journal of Medical Genetics. 58 (12), 715-718 (2015).</li><li>Shashi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+Truncating+Variants+in+ASXL2+Are+Associated+with+a+Unique+and+Recognizable+Clinical+Phenotype.">De Novo Truncating Variants in ASXL2 Are Associated with a Unique and Recognizable Clinical Phenotype.</a> American Journal of Human Genetics. 100 (1), 179 (2017).</li><li>Chen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+589,306+genomes+identifies+individuals+resilient+to+severe+Mendelian+childhood+diseases.">Analysis of 589,306 genomes identifies individuals resilient to severe Mendelian childhood diseases.</a> Nature Biotechnology. 34 (5), 531-538 (2016).</li><li>Halvorsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+mutations+in+early-onset+genetic+diseases.">Mosaic mutations in early-onset genetic diseases.</a> Genetics in Medicine. 18 (7), 746-749 (2016).</li><li>Kohler, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Phenotype+Ontology+in+2017.">The Human Phenotype Ontology in 2017.</a> Nucleic Acids Research. 45 (1), 865-876 (2017).</li><li>Rentzsch, P., Witten, D., Cooper, G. M., Shendure, J., Kircher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CADD:+predicting+the+deleteriousness+of+variants+throughout+the+human+genome.">CADD: predicting the deleteriousness of variants throughout the human genome.</a> Nucleic Acids Research. 47 (1), 886-894 (2019).</li><li>Sobreira, N., Schiettecatte, F., Valle, D., Hamosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GeneMatcher:+a+matching+tool+for+connecting+investigators+with+an+interest+in+the+same+gene.">GeneMatcher: a matching tool for connecting investigators with an interest in the same gene.</a> Human Mutation. 36 (10), 928-930 (2015).</li><li>Sobreira, N. L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matchmaker+Exchange.">Matchmaker Exchange.</a> Current Protocols in Human Genetics. 95 (9), 31-39 (2017).</li><li>Harnish, M., Deal, S., Wangler, M., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+functional+study+of+disease-associated+rare+human+variants+using+Drosophila.">In vivo functional study of disease-associated rare human variants using Drosophila.</a> Journal of Visualized Experiments. , (2019).</li><li>Harrison, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+ClinVar+as+a+Resource+to+Support+Variant+Interpretation.">Using ClinVar as a Resource to Support Variant Interpretation.</a> Current Protocols in Human Genetics. 89, 11-18 (2016).</li><li>MacDonald, J. R., Ziman, R., Yuen, R. K., Feuk, L., Scherer, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Database+of+Genomic+Variants:+a+curated+collection+of+structural+variation+in+the+human+genome.">The Database of Genomic Variants: a curated collection of structural variation in the human genome.</a> Nucleic Acids Research. 42, 986-992 (2014).</li><li>Firth, H. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DECIPHER:+Database+of+Chromosomal+Imbalance+and+Phenotype+in+Humans+Using+Ensembl+Resources.">DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources.</a> American Journal of Human Genetics. 84 (4), 524-533 (2009).</li><li>Thurmond, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FlyBase+2.0:+the+next+generation.">FlyBase 2.0: the next generation.</a> Nucleic Acids Research. 47, 759-765 (2019).</li><li>Consortium, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+genomics.+The+Genotype-Tissue+Expression+(GTEx)+pilot+analysis:+multitissue+gene+regulation+in+humans.">Human genomics. The Genotype-Tissue Expression (GTEx) pilot analysis: multitissue gene regulation in humans.</a> Science. 348 (6235), 648-660 (2015).</li><li>Ponten, F., Jirstrom, K., Uhlen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Protein+Atlas--a+tool+for+pathology.">The Human Protein Atlas--a tool for pathology.</a> Journal of Pathology. 216 (4), 387-393 (2008).</li><li>The Gene Ontology, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Gene+Ontology+Resource:+20+years+and+still+GOing+strong.">The Gene Ontology Resource: 20 years and still GOing strong.</a> Nucleic Acids Research. , (2018).</li><li>Mungall, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Monarch+Initiative:+an+integrative+data+and+analytic+platform+connecting+phenotypes+to+genotypes+across+species.">The Monarch Initiative: an integrative data and analytic platform connecting phenotypes to genotypes across species.</a> Nucleic Acids Research. 45 (1), 712-722 (2017).</li><li>Meehan, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease+model+discovery+from+3,328+gene+knockouts+by+The+International+Mouse+Phenotyping+Consortium.">Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.</a> Nature Genetics. 49 (8), 1231-1238 (2017).</li><li>Katoh, K., Rozewicki, J., Yamada, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT+online+service:+multiple+sequence+alignment,+interactive+sequence+choice+and+visualization.">MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization.</a> Brief Bioinform. , (2017).</li><li>Sievers, F., Higgins, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustal+Omega+for+making+accurate+alignments+of+many+protein+sequences.">Clustal Omega for making accurate alignments of many protein sequences.</a> Protein Science. 27 (1), 135-145 (2018).</li><li>Yoon, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Nardilysin,+a+Mitochondrial+Co-chaperone+for+alpha-Ketoglutarate+Dehydrogenase,+Promotes+mTORC1+Activation+and+Neurodegeneration.">Loss of Nardilysin, a Mitochondrial Co-chaperone for alpha-Ketoglutarate Dehydrogenase, Promotes mTORC1 Activation and Neurodegeneration.</a> Neuron. 93 (1), 115-131 (2017).</li><li>Deal, S., Yamamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+novel+mechanisms+of+neurodegeneration+through+a+large-scale+forward+genetic+screen+in+Drosophila.">Unraveling novel mechanisms of neurodegeneration through a large-scale forward genetic screen in Drosophila.</a> Frontiers in Genetics. 9, (2019).</li><li>Matamoros, A. J., Baas, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules+in+health+and+degenerative+disease+of+the+nervous+system.">Microtubules in health and degenerative disease of the nervous system.</a> Brain Research Bulletin. 126, 217-225 (2016).</li><li>Theodosiou, A., Arhondakis, S., Baumann, M., Kossida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+scenarios+of+Notch+proteins.">Evolutionary scenarios of Notch proteins.</a> Molecular Biology and Evolution. 26 (7), 1631-1640 (2009).</li><li>Shayevitz, C., Cohen, O. S., Faraone, S. V., Glatt, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+re-review+of+the+association+between+the+NOTCH4+locus+and+schizophrenia.">A re-review of the association between the NOTCH4 locus and schizophrenia.</a> American Journal of Medical Genetics. Part B: Neuropsychiatric Genetics. 159 (5), 477-483 (2012).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+and+re-evaluation+of+an+association+between+the+NOTCH4+locus+and+schizophrenia.">A review and re-evaluation of an association between the NOTCH4 locus and schizophrenia.</a> American Journal of Medical Genetics. 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Critical steps in this protocol include the initial input (steps 1.1-1.3) and subsequent interpretation of the output. The most common reason why search results are negative is because of the many ways that a gene and/or variant can be described. While MARRVEL is updated on a scheduled basis, these updates may cause disconnects between the different databases that MARRVEL links to. Thus, the first step in troubleshooting is invariably checking to see if alternative names of the gene or variant will lead to a successful s...

The use of next-generation sequencing technology is expanding in both research and clinical genetic laboratories1. Whole-exome (WES) and whole-genome sequencing (WGS) analyses reveal numerous rare variants of unknown significance (VUS) in known disease-causing genes as well as variants in genes that are yet to be associated with a Mendelian disease (GUS: genes of uncertain significance). Presented with a list of genes and variants in a clinical sequence report, medical geneticists must manually visit multiple online resources to obtain more information to assess which variant may be responsible for a certain phenotype seen in the patient of interest. This process is time-consuming, and its efficacy is highly dependent on the expertise of the individual. Although several guideline papers have been published2,3, interpretation of WES and WGS requires manual curation since there is yet to be a standardized methodology for variant analysis. For the interpretation of VUS, knowledge on the previously reported genotype-phenotype relationship, mode of inheritance, and allele frequencies in the general population become valuable. In addition, knowledge on whether the variant affects a critical protein domain, or an evolutionarily conserved residue may increase or decrease the likelihood of pathogenicity. To gather all of this information, one typically needs to navigate through 10-20 human and model organism databases since the information is scattered through the World Wide Web.
Similarly, model organism scientists who work on specific genes and pathways are often interested in connecting their findings to human disease mechanisms and wish to take advantage of the knowledge that is being generated in the human genomics field. However, due to the rapid expansion and evolution of data sets regarding the human genome, it has been challenging to identify databases that provide useful information. In addition, since most model organism databases are designed for researchers who work with the specific organism on a daily basis, it is very difficult, for example, for a mouse researcher to search for specific information in a Drosophila database and vice versa. Similar to the variant interpretation searches performed by medical geneticists, identifying useful human and other model organism information is time-consuming and heavily dependent on the background of the model organism researcher. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration)4 is a tool designed for both groups of users to streamline their workflow.
MARRVEL (http://marrvel.org) was designed as a centralized search engine that collects data systematically in an efficient and consistent manner for clinicians and researchers. With information from 20 or more publicly available databases, this program allows users to quickly gather information and access a large number of human and model organism databases without reiterative searches. The search result pages also contain hyperlinks to the original sources of information, allowing individuals to access the raw data and gather additional information provided by the sources.
In contrast to many of the variant prioritization tools that require large sequencing data input in the form of VCF or BAM files and installations of often proprietary/commercial software, MARRVEL operates on any web-browser. It can be used at no cost and compatible with portable devices (e.g. smartphones, tablets) as long as one is connected to the internet. We chose this format since many clinicians and researchers typically need to search one or a few genes and variants at a time. Note that we are developing batch-download and API (application programming interface) features for MARRVEL to eventually allow users to curate hundreds of genes and variants at a time through customized query tools if necessary.
Due to the wide range of applications, in this protocol, we will describe a broadly encompassing approach on how to navigate through different datasets that MARRVEL displays. More targeted examples that are tailored towards specific users&#8217; needs will be described in Representative Results section. It is important to note that the output of MARRVEL still requires a certain level of background knowledge in either human genetics or model organisms to extract valuable information. We refer the readers to the table that lists primary papers that describe the function of each of the original databases that are curated by MARRVEL (Table 1). The following protocol is divided into three sections: (1) How to begin a search, (2) how to interpret MARRVEL human genetics outputs, and (3) how to make use of model organism data in MARRVEL. In the Representative Results section, more focused and specific approaches are described. MARRVEL is being actively updated so please refer to the current website&#8217;s FAQ page for details about data sources. We strongly recommend the users of MARRVEL to sign up in order to receive update notifications through the e-mail submission form at the bottom of the MARRVEL home page.

<table border="0" cellpadding="0" cellspacing="0" style="width:753px;" width="752">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:133px;">Human Genetics</td>
			<td dir="LTR" style="width:169px;">ClinVar</td>
			<td style="width:169px;">PMID: 29165669</td>
			<td dir="LTR" style="width:281px;">https://www.ncbi.nlm.nih.gov/clinvar/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:133px;">Human Genetics</td>
			<td dir="LTR" style="width:169px;">DECIPHER</td>
			<td style="width:169px;">PMID: 19344873&#160;</td>
			<td dir="LTR" style="width:281px;">https://decipher.sanger.ac.uk/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:133px;">Human Genetics</td>
			<td dir="LTR" style="width:169px;">DGV</td>
			<td style="width:169px;">PMID:&#160;24174537</td>
			<td dir="LTR" style="width:281px;">http://dgv.tcag.ca/dgv/app/home</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:133px;">Orthology Prediction</td>
			<td dir="LTR" style="width:169px;">DIOPT</td>
			<td style="width:169px;">PMID: 21880147&#160;</td>
			<td dir="LTR" style="width:281px;">https://www.flyrnai.org/cgi-bin/DRSC_orthologs.pl</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Human Gene/Transcript Nomenclature</td>
			<td dir="LTR" style="width:169px;">Ensembl</td>
			<td style="width:169px;">PMID: 29155950&#160;</td>
			<td dir="LTR" style="width:281px;">https://useast.ensembl.org/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:133px;">Human Genetics</td>
			<td dir="LTR" style="width:169px;">ExAC&#160;</td>
			<td style="width:169px;">PMID: 27535533</td>
			<td dir="LTR" style="width:281px;">http://exac.broadinstitute.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">FlyBase (Drosophila)</td>
			<td style="width:169px;">PMID:26467478</td>
			<td dir="LTR" style="width:281px;">http://flybase.org</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Model Organism Database Integration Tools</td>
			<td style="width:169px;">Gene2Function</td>
			<td style="width:169px;">PMID: 28663344</td>
			<td dir="LTR" style="width:281px;">http://www.gene2function.org/search/</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:133px;">Human Genetics</td>
			<td dir="LTR" style="width:169px;">Geno2MP</td>
			<td>N/A</td>
			<td dir="LTR" style="width:281px;">http://geno2mp.gs.washington.edu/Geno2MP/</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:133px;">Human Genetics</td>
			<td dir="LTR" style="width:169px;">gnomAD</td>
			<td style="width:169px;">PMID: 27535533</td>
			<td dir="LTR" style="width:281px;">http://gnomad.broadinstitute.org/</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:133px;">Gene Ontology</td>
			<td dir="LTR" style="width:169px;">GO Central</td>
			<td style="width:169px;">PMID: 10802651, 25428369&#160;</td>
			<td dir="LTR" style="width:281px;">http://www.geneontology.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Human Gene/Protein Expression</td>
			<td dir="LTR" style="width:169px;">GTEx</td>
			<td style="width:169px;">PMID: 29019975, 23715323&#160;</td>
			<td dir="LTR" style="width:281px;">https://gtexportal.org/home/</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:133px;">Human Gene Nomenclature</td>
			<td dir="LTR" style="width:169px;">HGNC</td>
			<td style="width:169px;">PMID: 27799471&#160;</td>
			<td dir="LTR" style="width:281px;">https://www.genenames.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">IMPC (mouse)</td>
			<td style="width:169px;">PMID: 27626380</td>
			<td dir="LTR" style="width:281px;">http://www.mousephenotype.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">MGI (mouse)</td>
			<td style="width:169px;">PMID:25348401</td>
			<td dir="LTR" style="width:281px;">http://www.informatics.jax.org/</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:133px;">Model Organism Database Integration Tools</td>
			<td dir="LTR" style="width:169px;">Monarch Initiative</td>
			<td style="width:169px;">PMID: 27899636</td>
			<td dir="LTR" style="width:281px;">https://monarchinitiative.org/</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:133px;">Human Variant Nomenclature</td>
			<td style="width:169px;">Mutalyzer</td>
			<td style="width:169px;">PMID: 18000842&#160;</td>
			<td dir="LTR" style="width:281px;">https://mutalyzer.nl/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:133px;">Human Genetics</td>
			<td dir="LTR" style="width:169px;">OMIM</td>
			<td style="width:169px;">PMID: 28654725</td>
			<td dir="LTR" style="width:281px;">https://omim.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">PomBase (fission yeast)</td>
			<td style="width:169px;">PMID:22039153</td>
			<td dir="LTR" style="width:281px;">https://www.pombase.org/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:133px;">Literature</td>
			<td dir="LTR" style="width:169px;">PubMed</td>
			<td style="width:169px;">N/A</td>
			<td>https://www.ncbi.nlm.nih.gov/pubmed/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">RGD (rat)</td>
			<td style="width:169px;">PMID:25355511</td>
			<td dir="LTR" style="width:281px;">https://rgd.mcw.edu/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">SGD (budding yeast)</td>
			<td style="width:169px;">PMID: 22110037</td>
			<td dir="LTR" style="width:281px;">https://www.yeastgenome.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Human Gene/Protein Expression</td>
			<td dir="LTR" style="width:169px;">The Human Protein Atlas</td>
			<td style="width:169px;">PMID: 21752111</td>
			<td dir="LTR" style="width:281px;">https://www.proteinatlas.org/</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">WormBase (C. elegans)</td>
			<td style="width:169px;">PMID:26578572</td>
			<td dir="LTR" style="width:281px;">http://wormbase.org</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:133px;">Primary Model Organism Databases</td>
			<td dir="LTR" style="width:169px;">ZFIN (zebrafish)</td>
			<td style="width:169px;">PMID:26097180</td>
			<td dir="LTR" style="width:281px;">https://zfin.org/</td>
		</tr>
	</tbody>
</table>

navigating marrvel, a web-based tool that integrates human genomics and model organism genetics information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Human geneticists and model organism scientists each use MARRVEL in distinct ways, each with different desired outcomes. Below are three vignettes of possible uses for MARRVEL.
Evaluating pathogenicity of a variant in a dominant disease 
Most of the users that visit MARRVEL use this website to analyze the likelihood that a rare human variant may cause a certain disease. For example, a missense (17:59477596 G&#62;A, p.R20Q) variant in TBX2 was found to segregate i...

Watch this Scientific Journal Video about Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information at JoVE.com

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

MARRVEL.org aggregates information that can provide an overview of the disease relevance of human genes, focusing mostly on rare genetic diseases. It also gathers biological information from model organism databases. MARRVEL can save a lot of time and effort.Without this tool, users would spend hours searching different databases to gather information that MARRVEL can obtain in seconds. MARRVEL is actively used by physicians and scientists all over the world. It facilitates diagnosis and can be a starting point for exploring more information for therapeutic studies.Although most of our users have a human genetics background, we have increasing numbers of users from a wide range of biomedical fields, including model organism researchers. For a human gene and variant-based search, open the MARRVEL home page. A search can be performed with gene information alone, variant information alone, or both gene and variant information.Confirm that the candidate gene names are listed below the input box with each character entry. If the search comes back negative, use the HGNC website to make sure that the gene symbol used is up to date. Next, enter a human variant information to how variants are displayed on the Exome Aggregation Consortium and Genome Aggregation Database, or use a transcript-based nomenclature according to the Human Genome Variation Society guidelines.For genomic location nomenclature, use the coordinates according to Homo sapiens genome assembly 19. To access the model organism data relative to the human gene of interest in MARRVEL, use the Gene Function Table to obtain each of the following for eight model organisms. As each gene name is hyperlinked to the gene pages on their respective model organism databases, click on the links to find out more about the phenotypic information and resources available for each model organism.For example, on FlyBase, there will be a list of all of the alleles that have been generated, their respective phenotypes, and the availability of each allele from public stock centers. In the Gene Function Table, click the PubMed links to go to a list of publications that relates to the gene of interest in each organism. To see the confidence level that the model organism gene is likely to be the ortholog of the human gene of interest, check the DIOPT column to obtain a measure of the number of ortholog prediction algorithms that the model organism gene is likely to be an ortholog of the human gene of interest.Uncheck the Show only best DIOPT score gene box to display all of the candidates that typically include paralogous genes and homologous genes that are not necessarily orthologs in each model organism. To determine the expression of the gene or protein of interest, check the Expression column for the list of tissues where the gene or protein of interest has been reported to be expressed in human or model organism databases. Human transcriptome data is obtained from Genotype-Tissue Expression, or GTEx, and human protein expression data is from the Human Protein Atlas.In the Expression column, some data will have a button with pop-up links, such as for human and fly data, that display the gene or protein expression pattern using a heat map. Other data are hyperlinked to their respective model organism databases pages for further exploration. To access the Gene Ontology terms, check the Molecular function, Cellular component, and Biological process columns.These data have been filtered by the experimental evidence codes and display Gene Ontology terms that are based on direct experimental evidence in each model organism. Use Monarch Initiative to navigate to the Phenogrid page for a specific human gene. A chart that provides a quick comparison between the phenotypes associated with the gene of interest to known human diseases and model organism mutants that have phenotypic overlaps will appear.If a mouse gene has a knockout mouse made or planned by the International Mouse Phenotyping Consortium, clicking IMPC will link to the page that details the phenotype of the knockout mouse and its availability from public stock centers. To obtain information on predicted protein domains of the human protein of interest, click the Human Gene Protein Domains box. The data is derived from DIOPT, which uses protein families and Conserved Domains Databases.A single residue may be annotated more than once due to some overlap in domains annotated in the two sources. Then use the Multiple Protein Alignment box to obtain the amino acid multiple alignment generated by DIOPT that uses multiple alignment program for amino acid or nucleotide sequences aligner for each protein. To highlight the amino acid of interest, scroll down to the bottom of the box and enter the amino acid numbers of interest.To begin a search in MARRVEL based on model organism genes, click Model Organisms Search and select the model organism of choice. Enter a model organism gene symbol, click on the gene symbol as the name is auto-completed, and click Search. If the search result is negative, check the official gene symbol that is used in model organism databases.If the search result is still negative, access DIOPT and the Orthology Predictions Search database to assess if there are no good predicted orthologs for the gene of interest. If the search is positive, check the DIOPT score and the best score from human gene to model organism column for the selection of the human gene. If the gene has a DIOPT score of three or more and the best score from human gene to model organism is marked Yes, it is likely that there is more than one gene that is orthologous to the model organism gene of interest.Click the MARRVEL it link to display the search results for each human ortholog candidate. In this case, there is only one candidate. To interpret a MARRVEL human genetics output for a gene and variant of interest, click Online Mendelian Inheritance in Man, or OMIM.Use the Human Gene Description box to obtain a short summary of what is known about the gene and gene product. Use the Gene-Phototype Relationships box to determine if the gene of interest is known to be a Mendelian disease-causing gene. This box will provide manually curated known diseases and other phenotype associations with the gene of interest.The Reported Alleles From OMIM box can be used to obtain a list of pathogenic variants curated by this database to see if the variant of interest has been curated in OMIM as pathogenic. In this example, a search for human TBX2 was performed to assess the known biological function of this gene and the potential functional impact of a specific missense variant in the gene found to segregate in a small family with a rare disease that involves the skeletal, cardiac, and immune systems. Here, the output for the TBX2 search can be seen.Based on the model organism information displayed in MARRVEL, it was quickly determined that this gene of interest is conserved from C.elegans and Drosophila to humans and that the amino acid of interest, arginine 20, is also highly conserved throughout evolution. Note that rat TBX2 does not align well in this region, likely due to the transcript that is used for the alignment. By looking at the Gene Ontology terms of the TBX2 orthologs within different species, it can be observed that TBX2 encodes a transcription factor that has been implicated in heart morphogenesis and other biological processes in mouse and zebrafish.As human TBX2 is expressed in the heart-based genotype tissue expression and protein atlases and patients with a TBX2 variant exhibit heart defects, it can be concluded that this variant is a good candidate to explore experimentally. Gene names may change over time, so it is important to confirm that you're using the latest official gene symbols based on human or model organism databases. Please watch our accompanying JoVE video article titled In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila"for further experimental studies that can be performed.MARRVEL has been supporting clinicians and researchers in facilitating disease diagnosis and mechanistic studies. We will continue to do so by adding new features to the program as we move forward. MARRVEL is continuously being upgraded, so we recommend that users read the What's New section to stay up to date about the new features that have been introduced.

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Research

Education

JoVE Journal

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Molecular Biology

Genetics

Unit 1

DNA, Cells, and Evolution

SCIENTISTS IN ACTION

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

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 ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since &#34;low&#34; or &#34;high&#34; HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...