Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey.
BACKGROUND
Extensive computational and database tools are available to mine genomic and genetic databases for model organisms, but little genomic data is available for many species of ecological or agricultural significance, especially those with large genomes. Genome surveys using conventional sequencing techniques are powerful, particularly for detecting sequences present in many copies per genome. However these methods are time-consuming and have potential drawbacks. High throughput 454 sequencing provides an alternative method by which much information can be gained quickly and cheaply from high-coverage surveys of genomic DNA.


RESULTS
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).


CONCLUSIONS
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.
Swaminathan
Kankshita
K
Department of Crop Sciences, University Of Illinois, Urbana, IL 61801, USA. kank@uiuc.edu <kank@uiuc.edu>
Varala
Kranthi
K
Hudson
Matthew E
ME
D030561:Databases, Nucleic Acid; D018628:Gene Dosage; D018745:Genome, Plant; D023281:Genomics / Q000379:methods*; D008722:Methods; D012091:Repetitive Sequences, Nucleic Acid; D017422:Sequence Analysis, DNA; D013025:Glycine max / Q000235:genetics*