BMC Genomics 2007, 8:427 http://www.biomedcentral.com/1471-2164/8/427 $ "" $ & ’("# $ ("" $ & ’"# ! )# ! rEFexiagadumsrpele2s of repeat reconstruction from assembled 454 Examples of repeat reconstruction from assembled 454 reads. (A) The complete rDNA coding region including genes for 18S, 5.8S and 25S rRNA and parts of the large inter- genic spacer (IGS) was reconstructed as a single contig (CL2Contig6). The graph shows the read depth (number of assembled reads) along the contig sequence. (B) Reconstruc- tion of the Ty3/gypsy retroelement Peabody, including most of its long terminal repeat (LTR) sequence and complete polypro- tein-coding region (gag-pol). (C) A novel Ty1/copia element Ps-copia-1/751, reconstructed from ten overlapping contigs. The region devoid of stop codons encoding gag-pol was identi- fied in frame +3. Yellow bars depict length and relative posi- tions of the overlapping contigs, stop codons are represented by red vertical lines. The contig with the highest genome representation was a 5,097 bp fragment of the LTR-retrotransposon Peabody [GenBank: AF083074]. This contig (CL1Contig2066) had a read depth corresponding to 11,000 copies/1C and was estimated to make up 1.3% of the pea genome. It included a complete internal retrotransposon region surrounded by parts of LTR sequences, which could be further extended by finding and aligning overlapping contigs (Fig. 2B). The internal region contained a gag-pol coding sequence (4,368 bp) devoid of stop codons. The highest read depth (193 reads, corresponding to about 25,000 copies/1C) was found for a 1,213 bp contig CL1Contig751 representing the LTR sequence of a novel Ty1/copia element designated Ps-copia-1/751. The ele- ment reconstruction from ten overlapping contigs resulted in identification of the complete LTR and most of the internal region including open reading frame encod- ing gag-pol polyprotein (Fig. 2C). In addition to highly repeated sequences it was also pos- sible to at least partially reconstruct less abundant repeats, many of which were novel to the pea genome. For exam- ple, an over 7 kb region of a MuDR-like DNA transposon, including 2 putative coding regions, could be recon- structed from 25 overlapping contigs. MuDR elements were estimated to occur in about 2,200 copies in the pea genome, and similar abundance was also found for another DNA transposon family, En/Spm, for which it was possible to reconstruct a 3 kb fragment of the trans- posase-coding region (not shown). The clustering and contig building procedure was also found useful for identifying novel tandemly repeated sequences. Assembling overlapping reads into longer con- tigs facilitated reconstruction of repeats with monomers exceeding the length of single reads and allowed their identification based on the tandem subrepeats present within the contigs. A number of contigs representing potential satellite repeats with monomers from 50 to 867 bp were identified; except for the previously described PisTR-B satellite [20], they all represented novel sequences. Fourteen of the most abundant repeats (Table 1) were used as probes for in situ hybridization on pea mitotic chromosomes in order to test if they have a genomic distribution typical for satellite DNA. Such hybridization patterns, consisting of signals concentrated into limited number of spots corresponding to long arrays of the satellite sequences, were observed for thirteen repeats, whereas only one produced dispersed signals (Fig. 3A–C and Table 1). The signals occurred mostly in (peri-) centromeric and terminal chromosome regions, and each repeat displayed a specific hybridization pattern. No typical centromeric satellite repeats were found, although the repeat TR-11 strongly labeled central centro- meric regions in five out of the seven chromosome pairs (Fig. 3B). Page 4 of 16 (page number not for citation purposes)