BMC Genomics 2007, 8:427 http://www.biomedcentral.com/1471-2164/8/427 should be covered on average by 7–8 sequence reads (1000/129 = 7.8) over their entire length. In order to test this assumption, we determined the representation of pre- viously characterized pea repeats in the 454 data by sequence similarity searches against a database of individ- ual sequence reads and calculated their average coverage by highly significant hits. As expected, low-copy or mod- erately repeated sequences, such as Zaba MITEs (50–500 copies/1C [22]), were represented by none or only a few hits. However, all 33 of the tested sequences with an abundance exceeding 1,000 copies/1C were well repre- sented in 454 data, and their coverage by sequence reads was proportional to their abundance in the genome. The copy numbers of individual repeats calculated from the frequency of their occurrence in 454 reads were in a good agreement with estimates based on other experimental data (Fig. 1 and Additional file 1). These findings prove that the 454 data are representative for highly repeated sequences and thus can be further used for investigation and comprehensive description of this fraction of the pea genome. Reconstruction of genomic repeats by assembling 454 reads The process of extracting repeat sequences from the set of 454 sequence reads was implemented using TGICL, a pro- # " ! % $ !"# rFCmeioeagnmdutsrpaelalyr1istonthoef tehseti mreapteastbcaosepdy nounmthbeirrsfdreqteuremn cinyeidn e4x5p4eri- Comparison of the repeat copy numbers determined experimentally to the estimates based on their fre- quency in 454 reads. The comparison was performed for a set of previously characterized repetitive elements [20–24] including fragments of retrotransposon coding regions (gag- pol) and long terminal repeats (LTR), tandem repeats (TR), MITEs, and dispersed repeats of unknown origin (other). Detailed description of the repeats and their copy number estimates are available as Additional file 1. Dashed lines mark the area where the two estimates differ less than twofold. gram package originally designed for clustering large EST datasets [25]. The procedure consisted of two steps: (i) clustering the reads based on their mutual similarities into groups of overlapping sequences, and (ii) assembling the reads within the clusters to get longer fragments (contigs) representing consensus sequences. Various clustering and assembly parameters were evaluated in order to get opti- mal performance with our dataset, which compared to ESTs differed in the short reading lengths and in their con- siderable sequence variability, reflecting the divergence between individual copies of repeated elements. While the clustering parameters allowed for grouping of rela- tively divergent sequences, the assembly process was more stringent, and thus typically generated multiple contigs from a single cluster. The clustering resulted in 22,445 clusters, which were composed from two to thousands of reads. The assembly phase then yielded 25,384 contigs ranging in lengths up to 8,214 bp. Individual contigs were assembled from two to 4,327 reads, and the total number of sequence reads assembled into contigs was 233,303 (73 %). The contigs were characterized by their read depth (RD), expressing the average number of reads assembled over individual positions within the contig consensus sequence, and by their genome representation (GR), calculated as RD mul- tiplied by the contig length. These values allowed us to rate the contig sequences based on their genomic copy numbers and proportion in the genome, respectively. Most contigs were short and composed of only a few reads, whereas 90% of assembled reads were assigned into a relatively small subset of 1,578 contigs with the highest GR, thus corresponding to highly repetitive sequences. Most repeats were represented as sets of overlapping con- tigs, the number of which was proportional to the sequence diversity of the repeat copies. Among the most important in terms of their genomic abundance were con- tigs that included coding sequences and conserved LTR regions of Ogre retrotransposons and other LTR-retroele- ments and of the satellite repeat PisTR-B (Additional file 2). Compared to the three previously sequenced pea Ogre elements [21], this set of Ogre-like contigs showed much higher sequence diversity, suggesting they represent sev- eral distinct Ogre subfamilies. The longest contig (CL2Contig6) represented a 8,214 bp fragment of the rDNA gene cluster, including the com- plete 18-5.8-25S gene sequences (5,820 bp) surrounded by 3' and 5' parts of large intergenic spacer (Fig. 2A). Com- parison to previously published partial pea rDNA sequences revealed its identity with a 1,723 bp fragment of the 18S rRNA gene [GenBank: U43011] and 99.5% similarity to a 620 bp sequence region including the 5.8S rRNA gene and both internal transcribed spacers [Gen- Bank: AY839340]. Page 3 of 16 (page number not for citation purposes)