Blakesley et al. BMC Genomics 2010, 11:21 http://www.biomedcentral.com/1471-2164/11/21 extensive sequence-finishing efforts, which involve a low-throughput, craftsman-like process carried out by highly-skilled personnel performing both customized computational and experimental procedures. Alterna- tively, genomic sequence assemblies can be partially refined to less rigorous quality standards at a fraction of the cost and effort, with the resulting sequences still being extremely valuable for numerous applications, including comparative analyses [5-7]. Anecdotal experi- ences to date in sequencing vertebrate genomes have clearly revealed that each species’ genome presents its own characteristic set of challenges. Meanwhile, draft (but not finished) whole-genome sequences have now been generated for a large number of mammalian and other vertebrate species [8,9], without clear plans for finishing these genome sequences. The NISC Comparative Sequencing Program [10] has been extensively involved in inter-species sequence com- parisons. Specifically, we have generated sequence data from many vertebrates in parallel, focusing our studies on targeted genomic regions (as opposed entire gen- omes). This work has involved isolating, mapping, and sequencing orthologous bacterial artificial chromosome (BAC) clones from the same well-defined 0.5-2 Mb regions [6,11] of multiple vertebrate genomes and sys- tematically comparing the resulting sequences [6,7,12-20]. Our collective efforts to date have resulted in the generation of sequence for over 11,000 BACs iso- lated from 75 vertebrate species. The largest fraction of these data was produced as part of the ENCODE project [21]. In generating these BAC sequences, we have paid con- siderable attention to the sequence-finishing process. We routinely refine all BAC-derived sequence assem- blies to produce ‘comparative-grade’ finished sequence [5], the quality specifications of which were specifically designed for inter-species sequence comparisons. Com- parative-grade finished sequence is produced by estab- lishing the order and orientation of sequence contigs greater than 2 kb in size, and then independently verify- ing the resulting sequence contig map by auxiliary data. Our studies have shown that the quality of comparative- grade finished sequence is very high, with residual gaps and errors mostly residing within repetitive sequences [5]. To date, a subset of our sequenced BACs (>925) have also been finished to the near-perfect standards established for sequencing the human genome [4], which we refer to as ‘human-grade’ finished sequence. Our efforts in sequencing the same orthologous geno- mic regions in many different species and in producing two ‘grades’ of finished sequence for a large subset of BACs provided us a unique opportunity to examine results from application of the same standardized pro- cess to a variety of genome sequences. Here, we report Page 2 of 15 a series of studies that reveal substantial differences in the effort required to finish shotgun-generated genome sequences from different vertebrates, reflecting signifi- cant architectural differences between orthologous regions of these vertebrate genomes. Results Sequence data sets The nature of our comparative genome mapping and sequencing program [6,11] has required us to routinely sequence the DNA from many vertebrates. Our data and the anecdotal experiences of others have consis- tently suggested marked differences among species with respect to the effort required to refine (i.e., finish) shot- gun-generated sequence assemblies, even for the same orthologous genomic region. To investigate these differ- ences in a more systematic fashion, we generated and analyzed two comparable sequence data sets. The first data set (Table 1) consists of the human- grade finished sequences generated for 541 BACs from 38 vertebrates, with all sequences orthologous to a 1.9- Mb genomic region (in human) encompassing the cystic fibrosis transmembrane conductance regulator gene (CFTR); this genomic segment corresponds to ENCODE pilot project region ENm001 [22,23]. The number of sequenced BACs and the total amount of sequence in the ENm001-derived multiple-BAC sequence assembly generated for each vertebrate are provided in Table 1. A species’ sequence was included in this data set if Pip- Maker-derived alignments [24] of that species’ BAC sequences together covered at least 75% of the human ENm001 reference sequence. Importantly, comparative- grade finished sequence was also available for all 541 sequenced BACs in this first data set. The second data set consists of the comparative-grade finished sequences generated for 2,031 BACs from 21 vertebrate species (Table 2), with each sequence ortho- logous to one of 14 ENCODE pilot project regions (Table 3). The number of sequenced BACs and the total amount of sequence in the individual BAC clone sequences generated for each species (Table 2) or ENCODE region (Table 3) are listed. To be included in this data set, more than 75% of each of the ENCODE pilot project regions (Table 3) needed to be covered by PipMaker-derived alignments of comparative-grade fin- ished sequence from that species (Table 2). Most of the BAC sequences in this second data set have not been refined to human-grade finished sequence. Because of insufficient sequence coverage, BAC sequences were not included in this second data set from three other ENCODE pilot project regions (ENr231, ENr232, and ENr333) that have unusual characteristics and were thus analyzed for illustrative purposes (e.g., see below); in these cases, the number of sequenced BACs (and total