Blakesley et al. BMC Genomics 2010, 11:21 http://www.biomedcentral.com/1471-2164/11/21 respective indices, with each set of reads then assembled using Velvet [36]; the resulting sequence contigs for each BAC were then assembled with the original Sanger-based shotgun reads using Phrap. Preliminary results showed that the Velvet-derived sequence assemblies differed among the 12 BACs; for example, contig ‘N50’ size varied from 0.2 to 7 kb, with the ‘largest contig’ per BAC ranging from 2.8 to 24 kb. Of the contig sequences (totalling 2.35 Mb) that aligned to the Sanger-derived consensus sequences, there were few, if any, mismatches; thus, the quality of the Genome Analyzer-derived assembled sequence was extremely high. Combining the Sanger- and Genome Analyzer-derived shotgun sequence data simplified some of the sequence- finishing process, but did not solve the most vexing fin- ishing problems. For example, in some cases, the use of Velvet-assembled contigs helped to extend sequence from contig ends, provided complementary strand data, and resolved miscellaneous ambiguities (thereby elimi- nating the need to generate additional sequence reads during the finishing process). In fact, adding Genome Analyzer-derived data to the Sanger-derived assemblies reduced the total number of contigs for the 10 proble- matic BACs; for five BACs, a few of the uncaptured gaps were closed. However, new problems sometimes arose with the addition of Velvet-assembled contigs; for example, in several instances, new misassemblies formed where none existed in the Sanger-derived assembly. Most disappointing was the fact that many troublesome gaps in the Sanger-derived assemblies, which had been extremely problematic (and expensive) to close during human-grade sequence finishing, remained unchanged following the incorporation of Velvet-assembled contigs. Although some small contigs were found to partially fill these gaps, much of the missing sequence had no matching Genome Analyzer-derived sequence. These results point out the biases and limitations of both sequencing methods that utilize DNA polymerase. Discussion and conclusions The refinement of sequence assemblies generated from shotgun reads (i.e., sequence finishing) is a multi-step procedure that is influenced by various features of the starting template. Some of these features can present major problems during the sequence-finishing process; the most common being gaps (missing sequence) of var- ious types and characteristics, and sequence misassem- blies. Here, we present some representative findings based on our experience in finishing in a standardized fashion the sequence of over 11,000 BACs from 75 dif- ferent vertebrate species. This unique data resource allows for a rigorous assessment of the requirements for finishing sequence of diverse origins; the current study reveals substantial differences in the effort needed to Page 12 of 15 finish the sequence from different genomic regions and, in particular, from different species. While our experience provides the opportunity to gen- erate quantitative summaries about the comparative grade-finished assemblies of shotgun sequence from var- ious sources (e.g., Figures 1, 2 and 3), it also allows gen- eral qualitative conclusions to be drawn. For example, we have found that assembled shotgun sequence from: (1) rabbit and platypus sequences contain a higher than average number of SINEs and LINEs in and around regions associated with gaps, making the design of unique PCR primers for capturing the missing DNA impossible; (2) owl monkey, colobus monkey and tenrec sequence assemblies are often associated with regions of up to 2 kb that are biased with respect to the strand from which the shotgun reads were derived; (3) shrew and platypus are associated with a high frequency of large, uncaptured gaps; (4) dog and shrew are associated with gaps whose underlying sequence has a high GC content; and (5) baboon is associated with frequent misassemblies. Especially troublesome was the finding that hedgehog BACs are often associated with large deletions (50-100 kb) that occur during culturing of the clones prior to DNA extraction. In addition to the conclusions derived from analyzing the data sets reported here, we have gained considerable experience finishing sequences from other vertebrates. Based on this experience, we have found that: (1) little brown bat sequence assemblies are associated with numerous problematic dinucleotide repeats and a high frequency of uncaptured gaps whose underlying sequence has a high GC content; (2) opossum sequences are associated with large stretches of dinucleo- tide repeats at contig ends, often causing non-contiguous sequences to be incorrectly joined during assembly; and (3) echidna assemblies are associated with a high fre- quency of large, uncaptured gaps and with numerous problematic dinucleotide repeats. Interestingly, such fea- tures of the assembled sequence actually point to charac- teristic differences in genomic architecture among species. The insights gained from examining the underlying causes of sequence-finishing problems have allowed us to develop approaches to improve the finishing process, in some cases through proactive steps implemented in a species-specific fashion. For example, changing the host strain used for propagating shotgun subclones can improve the representation of certain sequences and reduce problems associated with sequence gaps. The use of different assembly programs can in some cases help to reduce the frequency and severity of misassembled sequences. Finally, relevant to recent technological advances, supplementing Sanger-derived shotgun reads with sequence data generated with a ‘next-generation’ DNA sequencing platform (e.g., an Illumina Genome