Blakesley et al. BMC Genomics 2010, 11:21 http://www.biomedcentral.com/1471-2164/11/21 The impact of contig number is most pronounced during the steps involved in establishing contig order and orientation, a key part of the finishing process. One approach involves comparing in silico- and laboratory- generated restriction enzyme digestion-based finger- prints of a BAC. When the assembled sequence is distributed among numerous contigs, in silico-deduced restriction fragments become artificially broken, making it is nearly impossible to derive an accurate restriction enzyme digestion-based fingerprint from the many com- binatorial possibilities. A second approach for ordering and orienting contigs involves aligning each contig to an orthologous reference sequence. Once again, interpret- ing the alignments of multiple small contigs in an assembled sequence is far more difficult than just a few large contigs (especially for sequences from species more distantly related to the reference sequence). For these reasons, the number of gaps in an assembled sequence is a critical parameter for the sequence-finishing process. We analyzed the first data set for the presence of gaps in the comparative-grade finished sequence of the 541 BACs. Specifically, for each of the 38 species listed in Table 1, we aligned the comparative-grade finished sequence of each BAC to the corresponding human- grade finished sequence for that species using the pro- gram Cross Match [28]. The resulting alignments were manually examined and corrected when contigs in the comparative-grade finished sequence had an ambiguous endpoint and/or order in the alignment. From the final alignments, we then catalogued the location, size, and sequence composition of all gaps in the comparative- grade finished sequence; further, the number of shotgun- library subclones spanning each gap (as detected by establishing the locations of all sequence reads in the human-grade finished sequence) was also determined. Across the 73.2 Mb of total sequence in the first data set (Table 1), we identified 1,528 gaps, corresponding to roughly 23 gaps per Mb. Notably, we found an identical fre- quency of gaps in an earlier study that involved analyzing the comparative-grade finished sequence of 116 vertebrate BACs [5]. Those 1,528 gaps together account for 1.1 Mb (or 1.5%) of the total sequence, and range in size from 1 bp to greater than 15 kb, with a median size of 250 bp; roughly a third (506) of the gaps are 500 bp or larger. Examination of the simple aggregate statistics above fails to reveal the striking differences between individual species with respect to gaps in their assembled genome sequences. For example, the amount of sequence resid- ing within gaps varied more than 15-fold among species (Figure 1A). For species like black lemur, tetraodon, and guinea pig, gap sizes are relatively small (~ 4 kb per Mb), whereas the gaps in shrew and platypus sequence correspond to an average of 36 and 58 kb per Mb, respectively. In the case of gap frequency, variation is Page 5 of 15 less pronounced, i.e., less than fourfold for all but three of the 38 species; in fact, gap frequency ranged from 17 to 23 per Mb for over half of the species, near the over- all median value of 20 gaps per Mb (Figure 1B). At the extremes for gap frequency are: (1) at the low end, black lemur and galago, with an average of 4 and 10 gaps per Mb, respectively; and (2) at the high end, platy- pus and torafugu, with an average of 42 and 48 gaps per Mb, respectively. A third characteristic, median gap size (Figure 1C), varies dramatically–from 67 bp for tetrao- don to 523 bp for horseshoe bat; platypus is again at the high end, with a median gap size of 477 bp. An overall comparison of frequency and size of sequence gaps indicates that some species’ sequences (e.g., guinea pig and galago) have below average values; the finishing of these sequences is therefore more straightforward. Chicken sequences are intermediate, with a high frequency of relatively small gaps. Finally, at the extreme of difficulty are platypus sequences, which have substantially more gaps of larger size (compared to the overall averages). Characteristics of sequences in gaps The characteristics of the sequence within and immedi- ately adjacent to a gap greatly influence the difficulty associated with filling that gap during sequence finish- ing. We thus examined the GC and repeat content of the sequence residing within gaps in the first data set, comparing those values to the overall averages for the total sequence generated for each species. The results for the 19 species with the greatest differences between gap sequence and total sequence are shown in Figure 2. For many species, the GC content of the gap sequence is only marginally different from the GC content of the total sequence (Figure 2A). For some species where large differences are noted (e.g., guinea pig, marmoset, and chicken), we have not found human-grade sequence finishing to be particularly difficult; the application of standard finishing strategies involving alternate sequen- cing reaction chemistry (e.g., 80:20 BigDye:dGTP BigDye or dGTP BigDye supplemented with SeqRxA) generally permits the generation of the missing sequence with minimal effort. However, this is not the case for other species. For example, the gaps in dog sequence average 57% GC content, considerably higher than the 40% GC content of the total sequence. One particular dog BAC sequence [GenBank:AC090445] contains a stretch of 2,100 bp with a GC content of 83%; an embedded gap of ~ 250 bp (85% GC content overall) was refractory to most routine gap-filling efforts and ultimately required an extraordinary finishing effort to close. Several other dog BAC sequences contain at least one gap that pre- sented similar challenges during finishing. Interestingly, such high GC-content regions in dog sequence gaps often reside near CpG islands.