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Questions and Answers (continued)

11) How to find BACs for a particular human gene (example "FUS")

How and where to we find this information?

There are different routes to find clone ID's and we will first explain one of these, which makes use of the University of California at Santa Cruz (UCSC) Genome Browser (A). Other routes might include the recently expanded (late 2008) NCBI CloneFinder program (B). The UCSC and NCBI links will open the Human Genome Browser and the Human Clone Finder program, respectively. If you're interested in a different species, then change the species name for the Browser/CloneFinder, and follow a somewhat similar procedure.

A) The following text applies only to the UCSC Browser. Type the gene name in the [position or search term] window to replace the default text. In this case, type FUS (as an example human gene).  Click the "Submit" button. A new page with many search results with data relevant to "FUS" appears. Make your best selection based on your knowledge of the FUS gene, for instance click the link for the RefSeq entry for [FUS at chr16:31098954-31110600] or the one linked to [Uncharacterized Protein FUS] (under UCSC Genes) [FUS (uc010caj.1) at chr16:31107147-31109220].  Once you click on these links, you will get a map encompassing the genomic features of a 2,074 bp chromosome segment of chromosome 16p11.2. You can change the display: zoom in or zoom out, display additional genomic features, etc. You may also see a series of horizontal lines under the heading of "BAC End Pairs" and horizontal lines have arrow heads pointing to the left or the right. This "arrow" polarity indicates the relative orientations of insert and (BAC)vector sequences. If you don't see any horizontal lines and a "BAC End Pairs" heading, then you will need to activate the "BAC End Pair" display option on the Genome Browser to "Full" and hit the "Refresh" button within the UCSC Genome Browser.  If you see the BACs, how do you know the size and boundaries of the BAC insert sequence? You can either zoom out until you see the two insert ends displayed, or you can click on one of the horizontal "BAC" lines. When you click, a new page opens with many links to the related BAC-insert End Sequences ("BES"). You may also see a horizontal "Green" bar indicating clones (mostly BACs) which have been mapped by Fluorescent In Situ Hybridization (and other approaches) to this location. All of the BACs displayed will either be from the RPCI-11 BAC library (created in our laboratory (previously at Roswell Park Cancer Institute) or from the CTD (Caltech-D BAC library). We only distribute (with few exceptions) the RP11 BAC clones. In addition to BACs, you may also see fosmid clones (names starting with "G248") and these are also distributed from our BACPAC Resources Center. The fosmid are part of the WIBR-2 human fosmid library created at MIT. If you don't see the fosmids displayed within the UCSC Genome Browser, then make sure to activate the "Fosmid_end Pairs" option to "Full", followed by "Refresh". How much information is available for any of these BACs or fosmids? All of these have only been sequenced very partially: at most a few hundred base pairs from both insert ends. The sequence pairs have been mapped to the assembled human genome and have been approved if the orientation and distance of the sequence pair is compatible with the size of a typical BAC or fosmid clone.

You may be lucky to find a BAC clone which has been completely sequenced AND includes the complete locus. How does one identify such a completely sequenced BAC? Check on the dashboard of options (below the UCSC map) if the "Assembly" option has been activated to "Full". If necessary, activate this option and "Refresh" the browser. Then, inspect the top of the map and look for horizontal brown bars under the heading "Assembly from Fragments". The brown bars represent sequence assemblies obtained from shotgun-sequenced clones, in most cases BAC clones and 75% are derived from the "RP11" a.k.a "RPCI-11" BAC library. All of the clone-derived assemblies are labeled by their NCBI sequence accession numbers, for instance for the Fus gene): "AC009088.9". Inspect the annotations for NCBI sequence accession file and you will find the name of the clone (in this case "RP11-388M20"). Please note: the finished sequences are sometimes only a part of the BAC and the BAC may be bigger! Why? Because an overlapping BAC clone may also have been sequenced and only one sequence of the overlap was finished and used to created the human reference sequence. Be also aware that the clone names in the sequence files are not always standard names in compliance with the NCBI Nomenclature rules . The reason for the non-compliance is historical: some clones were sequenced before the Nomenclature rules were established. If you want to order a "non-compliant" clone, then translate the name into standard nomenclature before ordering the clone through our electronic shopping cart.

B) An additional option for finding clones for many human libraries became available late 2008. This involves the use of the NCBI CloneFinder program . The program is self-explanatory. Please realize that the number of libraries and options may be more abundant than needed. Please activate only the RPCI-11 (RP11, BAC), CHORI-17 (CH17, BAC) and W12 (fosmid) search options if you want clones available through our BACPAC Resources Center. After specifying BAC libraries and the search feature (region spanning from “Fus to Fus”), you may obtain the following weblink . If this is done for the “Fus” gene example, you will obtain data indicating BAC clones CH17-103K03 and RP11-112L3, and also 7 fosmid clones. Please remove the non-compliant "zero" from the CH17 clone name before ordering online from our BACPAC Resources Center: CH17-103K3 is the correct name.

Following the instructions under (A) or (B), you will likely have found some or many BACs and fosmids containing the FUS locus (our example). Most of the displayed BACs have a name starting with "RP" or "CH17". The "RP11" and "CH17" BAC libraries were made in our BACPAC laboratory and a more extensive description can be found on the corresponding pages in our BACPAC library browser (look for the full library names: RPCI-11 and CHORI-17 ). You will discover that the first library is derived from an anonymous "diploid" human donor for the Human Genome Project. The second library is from a "haploid" human DNA source, which has been sequenced at the Washington University Sequencing Center. You might eventually be able to obtain the complete sequence equivalent to CH17 BACs through data mining (retrieval of the corresponding sequence from the haploid sequence assembly). The WIBR-2 fosmid library was created at MIT as to support the original human genome assembly. The fosmids can also be ordered from our BACPAC Resources Center.

Please let us know if any of the links in this answer are no longer working, and we will update the text.

 

 

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