AUTOMATED FINGERPRINTING OF BAC LIBRARIES FOR THE CONSTRUCTION OF PHYSICAL MAPS OF LARGE GENOMES

1. AUTOMATED FINGERPRINTING OF BAC LIBRARIES FOR THE CONSTRUCTION OF PHYSICAL MAPS OF LARGE GENOMES M.-C. Luo1 , C. Thomas1 , Z. Xu2 , H.-B. Zhang2 , M. Malandro3 , M. Morgante4 , P., E. McGuire5 , J. Dvorak1 1 Department of Agronomy and Range Sciences, University of California, Davis, CA 95616, USA; 2 Department of Soil and Crop Sciences and Institute for Plant Genomics and Biotechnology, 2123 TAMUS, Texas A & M University, College Station, TX 77843-2123, USA; 3 Sagres Discovery, 2795 Second Street Suite 400, Davis, CA 95616, USA;4 E. L. duPont de Nemours and Co., DuPont Crop Genetics, Newark, Delaware 19714, USA; 5 Genetic Resources Conservation Program, University of California, One Shields Ave., Davis CA 95616 USA INTRODUCTION Physical maps of genomes based on ordering large-insert clones are valuable tools for genomic research. EST-integrated physical maps of large genomes, such as those of many economically important plants and animals, are a low-cost alternative to genome sequencing. The development of physical maps necessitates contiging large-insert clones. Contigs can be produced by end-sequencing of clones (Ventner, 1996) or by the development of a restriction digest "fingerprints". The latter can be generated by restriction digestion of clones and sizing of the restriction fragments by agarose gel electrophoresis (Coulson, 1986), polyacrylamide gel electrophoresis (Chang, 2001), or capillary electrophoresis. The fragment sizing can be automated by employing gel-based DNA sequencers (Ding, 1999) or capillary-based DNA sequencers. The former automation strategy was used to contig maize BACs (Faller, 2001). The greatest potential for high-throughput, automated fingerprinting is offered by capillary electrophoresis. The objective of this project is to construct the physical maps of Aegilops tauschii (4,000 Mbp/1C), the donor of the wheat D genome. To accomplish this,A minimum of 200,000 BAC clones need to be fingerprintedfor genome of this size. To accomplish this daunting task, the entire process must be automated. We describe here a basic strategy and its validation for the high-throughput fingerprinting of Ae. tauschii BACs by capillary electrophoresis. RESULTS A typical fingerprinting electropherogram produced by ABI 3100 is shown in Fig. 2. The peaks represent fluorescence of fluorescent dye labeled restriction fragments.To distinguish the real peaks from noise caused by residual partial BAC digests and bacterial DNA contamination, the following computer algorithm was designed and applied automatically to each profile. The tallest 2 peaks in the profile were disregarded and the heights of the ten highest remaining peaks were averaged. Only peaks with a height within 65% of this mean were considered real by the computer. The agreement between the numbers of expected and observed fragments in the fingerprinting profiles for BACs #1 and #2 are summarized in Tables 1 and 2. The overall success rate in detecting DNA fragments in the rangefrom 80 bp to 500 bp in the fingerprintwas 99% for BAC #1 and 97% for BAC #2 (Tables 1 and 2). The nucleotide sequences of the two BACs predict 13 of a total of 209 restriction fragments in size 80 to 500 bp are shared. A total of 11 of these fragments differing from each other by less than ± 0.2 nucleotides were found in the profiles of the two BACs (Table 3). Table 1. Comparison of the numbers of restriction fragments recorded by the computer program in the fingerprint profiles and those predicted from the nucleotide sequence of BAC #1 MATERIALS AND METHODS The high-throughput BAC fingerprinting strategy is shown schematically in Fig. 1. BAC DNAs were isolated with either Autogen 850 or Autogen 960. The latter is designed to isolate BAC DNAs in a 96-well block format, with a throughput of 2,400 DNAs/day. To produce fingerprints, BAC DNAs were digested with four 6-bp recognizing restriction enzymes creating 5’ overhangs with A (Eco RI), T (Xho I), C (Xba I) and G (BamHI) on the overhanging strand adjacent to the 3’ends. Each of the four restriction sites was labeled with different fluorescent dye by the SNaPshotTM kit (Applied Biosystems). To produce a population of labeled restriction fragments in the range of 80 to 500 bp, the fragments were digested with HaeIII restriction enzyme which produces a blunt end cut. Digestion and labeling were performed in three separate steps which necessitates the use of robotics (Tecan Genesis RSP 150) to achieve a high-throughput. Labeled restriction fragments were sized on an ABI 3100 capillary genetic analyzerutilizing the 36 cm capillaries with a throughput of about 500 DNAs/day. To test the fidelity of the fingerprinting method, two fully sequenced Triticum monococcum BACs #1 and #2 (107 and 121 kb long, respectively, with a 20.6 kb overlap), kindly provided by J. Dubcovsky of University of California, Davis, were fingerprinted and the fragments observed in the profiles were compared with the restriction fragments expected on the basis of the nucleotide sequence. Table 2. Comparison of the numbers of restriction fragments recorded by the computer program in the fingerprint profiles and those predicted from the nucleotide sequence of BAC #2 Table 3. The numbers of restriction fragments shared by BACs #1 and #2 predicted from their nucleotide sequence and those found to be shared because of their size being identical within ± 0.2 nucleotide in the profiles of BAC #1 and BAC #2. Fig. 1. The scheme of sample flow through the high-throughput fingerprint procedure. Fig. 2. A fingerprint of BAC #2 with XhoI (the remaining three electropherograms are not shown). The sizing of fragments by the instrument labeled by SNaPshot is only approximate (within 8 bp of the absolute size) relative to LIZ 500 size standards (called "relative size"). Note that all 26 expepectedrestriction fragments predicted by the DNA sequence are present in the profile. REFERENCES Chang Y.L., Q.Z. Tao, C. Scheuring, K.J. Ding, K. Meksem & H.B. Zhang, 2001. An integrated map of Arabidopsis thaliana for functional analysis of its genome sequence. Genetics, 159: 1231-1242. Coulson A., J.S. Sulston, S. Brenner & J. Karn, 1986. Toward a physical map of the genome of the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 83: 7821-7825. Ding Y., M.D. Johnson, R. Colayco, Y.J. Chen, J. Melnyk, H. Schmitt & H. Shizuya, 1999. Contig assembly of bacterial artificial chromosome clones through multiplexed fluorescence-labeled fingerprinting. Genomics, 56: 237-246. Faller M.L., K.A. Fengler, T. Dam, D. Leyva, M. Dolan, S.V. Tingy & M. Morgante, 2001. Integrating, genetic and physical maps for positional cloning in corn:ESTs, SNPs, and BACs. PAG IX, San Diego, 38. Ventner J.C., H.O. Smith & L. Hood, 1996. A new strategy for genome sequencing. Nature, 381: 364-366. • CONCLUSIONS • A high-throughput, fully automated BAC fingerprinting procedure has been developed facilitating fingerprinting of up to 2,000 BACs/day (with one AutoGen 960 and four ABI 3100 Instruments). With this rate, the Ae. tasuchii genome can be fingerprinted in 100 working days. • The fidelity of this fully automated fingerprinting procedure utilizing the SNaPshot kit is 97% or higher. • The sensitivity of the procedurefor detecting restriction fragments shared by two BACs to contig them is estimated to be 85%. Ifan overlap of 90% of restriction fragments by two BACs is usually used to generate a BAC contig, this level of sensitivity is far more than adequate.