Bosi et al. BMC Genomics 2011, 12:403 http://www.biomedcentral.com/1471-2164/12/403 PAGs). It must be stated clear that this kind of strategy has only an explorative purpose and might be strongly influenced by the present content of public databases that, undoubtedly, represents just a glimpse of the real biodiver- sity present in nature. For this reason, the reliability of the developed approach was firstly revealed by a test on a set of 1000 likely chromosomal native sequences retrieved by Cortez et al. [10] from a set of 119 bacterial and archaeal chromosomes. Indeed, results of this preliminar screening showed that almost 90% of the probed sequences were correctly annotated, i.e. resulted to possess a putative chromosome origin. Furthermore, we sought to test the implemented strategy on a set of sequences of plasmid and phage origin. However, as already pointed out, in these cases a set of “core” sequences is very difficult (if not impossible) to be retrieved. Hence, the previously described pipeline was applied on two distinct randomly assembled datasets of viral and plasmids sequences, embedding 1000 sequences each. Results similar to those obtained with (likely) chromosome native sequences were obtained (84% and 82% of “correct” identifications in the case of plasmdis and phages, respectively) thus suggesting that, in most cases, the implemented strategy is able to detect the correct source molecule of a given sequence. Applying the described strategy to our PAGs dataset revealed that, as shown in Figure 7, most of the identified Figure 7 PAGs origin. Pie chart showing the most likely source molecule of retrieved PAGs. Und. stands for “Undetermined “ (see text for details). Page 8 of 12 PAGs (almost 75%) are of likely plasmid origin. On the contrary, chromosome and phage sequences appear to be much rarer, being represented by 5.7% and 4.8% of PAGs, respectively. Remarkably, almost 13% of all the probed PAGs (1087), did not retrieve a clear corresponding match in the plasmid, chromosome and phage databases and, for this reason, their origin remained “undetermined” (Figure 7). Finally, less than 1% of the PAGs did not retrieve any match in any of the databases during our BLAST probings and were labelled as “Not Found” in Fig- ure 7. Again, also sequences retrieved with lower CI thresholds showed the same hypothetical origin, with likely plasmid orgin sequences being largely over-repre- sented (73.8%, 72.9% and 73.5% for 70%, 80% and 90% CI thresholds, respectively). Taken together these results indi- cate that most of the PAGs are likely to appear only in plasmids rather than begin shared among different types of informative molecules. We speculate here that most of retrieved PAGs likely derive from plasmid-plasmid gene exchanges and that, vice versa, integrations following virus-plasmid and/or chromosome-plasmid gene exchange appear to be less frequent. Remarkably, this finding fits with previous analyses [40] on the structure of the overall DNA-exchange network and that suggested that DNA families are mostly exchanged among the same type of DNA carriers (i.e. plasmids, phages or chromosomes). Our analyses indicate that this might hold true (at least) in the case of plasmid molecules. Clusters of PAGs Chromosomal atypical genes are often organized in (more or less) tight clusters. These chromosomal regions, named genomic islands [10], embeds, for exam- ple, genes involved in pilus and fimbriae formation, lipo- polysaccharide biosynthesis or virulence and have been shown to have played a crucial role in microbial evolu- tion [41]. Similarly, PAGs might either stand alone or be embedded in (more or less compact) clusters on their source molecule representing, for example, suc- cessfully integrated transposons and/or integrons. Hence, we investigated this issue and evaluated if retrieved PAGs lied in clusters or were scattered throughout the corresponding plasmid. We estimated gene clustering at three different gene distance thresh- olds, that is 100, 200 and 300 bp. Results shown below refer to genes that are not separated by more than 200 bp (results for 100 and 300 bp are provided as Addi- tional file 8, together with clusters distribution retrieved at different CI thresholds). Overall we found that 1653 PAGs (on a total of 8065) are embedded in 677 clusters of different size (Table 1 and Figure 8). The complete list of all identified PAGs clusters, together with their corresponding organisms and GI codes, are provided as additional material (see Additional file 9). Most of them