Whole-genome hybridization studies have suggested that the nuclear genomes of 
accessions (natural strains) of Arabidopsis thaliana can differ by several 
percent of their sequence. To examine this variation, and as a first step in the 
1001 Genomes Project for this species, we produced 15- to 25-fold coverage in 
Illumina sequencing-by-synthesis (SBS) reads for the reference accession, Col-0, 
and two divergent strains, Bur-0 and Tsu-1. We aligned reads to the reference 
genome sequence to assess data quality metrics and to detect polymorphisms. 
Alignments revealed 823,325 unique single nucleotide polymorphisms (SNPs) and 
79,961 unique 1- to 3-bp indels in the divergent accessions at a specificity of 
>99%, and over 2000 potential errors in the reference genome sequence. We also 
identified >3.4 Mb of the Bur-0 and Tsu-1 genomes as being either extremely 
dissimilar, deleted, or duplicated relative to the reference genome. To obtain 
sequences for these regions, we incorporated the Velvet assembler into a 
targeted de novo assembly method. This approach yielded 10,921 high-confidence 
contigs that were anchored to flanking sequences and harbored indels as large as 
641 bp. Our methods are broadly applicable for polymorphism discovery in 
moderate to large genomes even at highly diverged loci, and we established by 
subsampling the Illumina SBS coverage depth required to inform a broad range of 
functional and evolutionary studies. Our pipeline for aligning reads and 
predicting SNPs and indels, SHORE, is available for download at 
http://1001genomes.org.