High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

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dc.contributor.authorGeorge, Philipen
dc.contributor.authorSharakhova, Maria V.en
dc.contributor.authorSharakhov, Igor V.en
dc.contributor.departmentEntomologyen
dc.date.accessioned2017-01-05T23:40:25Zen
dc.date.available2017-01-05T23:40:25Zen
dc.date.issued2012-06-01en
dc.description.abstractProjects to obtain whole-genome sequences for 10,000 vertebrate species and for 5,000 insect and related arthropod species are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquito species is currently being done using an Illumina platform. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented. Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosomebased genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations. Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.en
dc.description.versionPublished versionen
dc.format.extent? - ? (8) page(s)en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.3791/4007en
dc.identifier.issn1940-087Xen
dc.identifier.issue64en
dc.identifier.urihttp://hdl.handle.net/10919/73972en
dc.language.isoenen
dc.publisherJournal of Visualized Experimentsen
dc.relation.urihttp://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000209223100034&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=930d57c9ac61a043676db62af60056c1en
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivs 3.0 Unporteden
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/3.0/en
dc.subjectgeneticsen
dc.subjectissue 64en
dc.subjectentomologyen
dc.subjectmolecular biologyen
dc.subjectgenomicsen
dc.subjectautomationen
dc.subjectchromosomeen
dc.subjectgenomeen
dc.subjecthybridizationen
dc.subjectlabelingen
dc.subjectmappingen
dc.subjectmosquitoen
dc.titleHigh-throughput Physical Mapping of Chromosomes using Automated in situ Hybridizationen
dc.title.serialJove-Journal of Visualized Experimentsen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
pubs.organisational-group/Virginia Techen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/CALS T&R Facultyen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/Entomologyen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Faculty of Health Sciencesen

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