Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey

We sequenced 78 million base-pairs of randomly sheared soybean DNA which passed our quality criteria. Computational analysis of the survey sequences provided global information on the abundant repetitive sequences in soybean. The sequence was used to determine the copy number across regions of large genomic clones or contigs and discover higher-order structures within satellite repeats. We have created an annotated, online database of sequences present in multiple copies in the soybean genome. The low bias of pyrosequencing against repeat sequences is demonstrated by the overall composition of the survey data, which matches well with past estimates of repetitive DNA content obtained by DNA re-association kinetics (Cot analysis).This approach provides a potential aid to conventional or shotgun genome assembly, by allowing rapid assessment of copy number in any clone or clone-end sequence. In addition, we show that partial sequencing can provide access to partial protein-coding sequences.Genome sequencing has historically been accomplished by fragmenting genomic DNA, amplifying the fragments clonally using bacteria, and sequencing the amplified clones [1]. Although this method has improved to the extent that much larger genomes can be sequenced, and some of the intermediate cloning steps can be circumvented [2,3], practically all genome sequence until very recently has been generated by the Sanger method. Given the costs of Sanger-based genome sequencing and surveys, significant amounts of genomic information for most of the 129,293 eukaryotic species listed in the NCBI taxonomy database [4] are unlikely to be available for some time. Soybean [Glycine max (L.) Mer], which is the subject of this study, has an existing but incomplete genome project. However, many crop plants, plant pathogens, endangered species and species of evolutionary interest have little or no available genome data. Recently developed microbead technologies capable of sequencing hundreds of thousan