Bosi et al. BMC Genomics 2011, 12:403 http://www.biomedcentral.com/1471-2164/12/403 assembly of larger PAGs dataset, ranging from 14731 (with a CI of 90%) to almost 40000 (with a CI of 70%) (see Additional file 1). Identification of PAGs source molecules In order to identify the most likely source molecule of identified PAGS we developed a similarity-oriented computational pipeline according to which each of the identified PAG was used as a query for a BLAST [27] search against three different databases, each of which embedding 30000 sequences retrieved from NCBI plas- mid, phage and chromosome databases (see Methods), respectively. For each of the BLAST searches, only the best BLAST hit was considered, in order to reduce any possible bias due to the presence of closely related sequences in the database that would falsely increase the number of homologs for a given ORF. This strategy was repeated 1000 times for each PAG and, for each of the 1000 runs, new plasmid, chromosome and phage data- bases were assembled, randomly sampling 30000 sequences from the NCBI databases. Finally, the putative source molecule was identified according to the database (plasmid, phage or chromosome) that produced the highest number of best hits after 1000 BLAST probings. Statistics All statistical tests were performed with the R package [28]. All other statistical analyses were performed using in-house developed Perl scripts. Results and Discussion PAGs general feaures PAGs distribution We applied the computational pipeline described in Methods (Figure 1) to 1853 archaeal (51) and bacterial (1802) plasmids (128.569 ORFs in total) in order to explore their mosaic structure, using a confidence value of 95% (see Methods for details). In this way we were able to collect 8065 compositionally atypical ORFs, denominated PAGs (that is 6.2% of all the 128.569 encoded proteins, Table 1) distributed through 1354 plasmid molecules (73% of the entire dataset). The Table 1 PAGs general features N. of analyzed plasmids N. of analyzed sequences N. of retrieved PAGs Percentage of PAGs Average PAGs for plasmid PAGs in clusters (≥ 2 genes) Cluster of PAGs (≥ 2 genes) PAGs per clusters (on average) 1853 128.569 8065 6.2% 4.3 1653 677 2.4 Page 4 of 12 Figure 2 PAGs distribution. PAGs ditribution across the plasmids composing the dataset. remaining plasmids (519, 27% of the dataset) do not possess any atypical gene at all. The fasta format of PAGs sequences (including those retrieved at a CI of 70%, 80% and 90%) is available as Additional file 2. The analysis of the distribution of PAGs (that is the number of PAGs per plasmid) within the assembled plasmid dataset revealed that they are not evenly distributed (Figure 2), ranging from 59 (found in Methylobacterium extorquens AM1) to 1 PAG (342 plasmids). Overall the distribution of PAGs across the microbial dataset showed that a high number of plasmids possess a small number of PAGs (or do not possess any PAGs at all) whereas only a few of them possess higher number of atypical ORFs (Figure 2). Importantly, the same trend was observed with PAGs dataset retrieved at lower CI, i. e. 70%, 80% and 90% (Additional file 3). To find possible correlations between PAGs distribution and the taxonomy of their hosting cells, we evaluated the percentage of PAGs for each of the genera embedded in our dataset. For clarity purposes, in Figure 3 we report only results concerning those genera for which it was possible to retrieve at least 7 plasmids dur- ing dataset assembly procedures (see Methods); com- plete results are provided in Additional file 4. Overall, we found that PAGs number varies greatly among the different genera (Figure 3). Besides, since the taxonomi- cal distribution of PAGs might be strongly influenced by the overall number of sampled sequences from each genus, we evaluated the statistical significance of this distribution by comparing them with 10000 randomly assembled ones, obtained re-shuffling the 8065 PAGs within the entire plasmids dataset and counting the frac- tion of times each genus possessed a number of PAGs