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CHORI-276: Orangutan, Sumatran (F) (Pongo abelii) BAC Library |
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The CHORI-276 (Sumatran) orangutan BAC library has been constructed by Yuko Yoshonaga in Pieter de Jong’s laboratory at BACPAC Resources, Children’s Hospital Oakland Research Institute. The preparation of the library followed the cloning approach developed in our laboratory (Osoegawa et al., 1998). To obtain DNA, a request for a blood sample was submitted to Dr. Greely Stones from the Gladys Porter Zoo. After favorable review, and coinciding with a scheduled annual physical examn, a blood sample was provided from a female sumatran orangutan "Susie" (Studbook #1044; ISIS #71). Susie is the mother of the male orangutan "Segundo" used as the DNA source for the CHORI-253 BAC library. High-molecular-weight DNA was isolated from white blood cells by embedding in agarose. The embedded DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI Methylase and size fractionated by pulsed-field electrophoresis. DNA fragments from the appropriate size fraction were cloned into the pBACGK1.1 vector between the two EcoRI sites. The ligation products were transformed into DH10B (T1 resistant) electro-competent cells (Invitrogen). The library has been arrayed into 528 “384-well” microtiter dishes and has subsequently been gridded onto eleven 22x22cm nylon high-density filters for probe hybridization screening. Each hybridization membrane represents over 18,000 distinct BAC clones (48 dishes), stamped in duplicate. Library characterization was performed by Teresa Ren and Kazutoyo Osoegawa. BAC library construction was funded by NIH grant HG025323-03 under the auspices of the NIH-funded BAC Resource Network.Segment | 1 | 2 | All | Vector | pBACGK1.1 | pBACGK1.1 | pBACGK1.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | N/A | WBC | N/A | Plate Numbers | 1-288 | 289-528 | 1-528 | Plate Count | 288 | 240 | 528 | Empty Wells | 3583 (3.24%) | 3226 (3.5%) | N/A | Non-Recombinant Clones | N/A | N/A | N/A | Non-Insert Clones | Approx. 1216 (1.1%) | Approx. 2949 (3.2%) | N/A | Recombinant Clones | 105793 | 85985 | 202752 | Average Insert Size | 177 Kbp | 176 Kbp | N/A | Genomic Coverage | N/A | N/A | N/A |
Click here for a legend of the previous tables.
The library is available for screening using hybridization filters, which can be ordered per complete set of 11 membranes (price per set is 11 times $200.00) or per segment. Single clones are being distributed in LB-agarose stab cultures. See ordering instructionsif you want to place an order. For hybridization membranes, please contact BACPAC Resources ([email protected]).
Academic or commercial users interested in a copy of the BAC library should contact Pieter J. de Jong ([email protected], fax: (510) 450-7924).
This BAC library was created from the same individual (Susie) sequenced as part of for the Orangutan Genome Assembly. The genome of a female Sumatran orangutan was sequenced using a whole-genome shotgun (WGS) strategy. The assembly provides 5.5-fold coverage on average across 3.08 gigabases (Gb) of ordered and oriented sequence. The accuracy of the WGS assembly was assessed by several metrics, including comparison to 17 megabases (Mb) of finished bacterial artificial chromosome (BAC) sequences (see the Nature manuscript by Locke et al., 2011. The long-range continuiety of the orangutan WGS assembly was also facilitated through the use of paired BAC-end Sequences ("BES") from the CHORI-276 BAC library. The 283,265 BES records can be retrieved from the NCBI Trace Archive. The ~4 million sequence reads from small-insert ("shotgun") plasmid libraries derived from CHORI-276 BACs can also be found in the NCBI Trace Archive.
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