Blakesley et al. BMC Genomics 2010, 11:21 http://www.biomedcentral.com/1471-2164/11/21 R E S E A R C H A R T I C L E Open Access Effort required to finish shotgun-generated genome sequences differs significantly among vertebrates Robert W Blakesley1,2, Nancy F Hansen2, Jyoti Gupta1, Jennifer C McDowell1, Baishali Maskeri1, Beatrice B Barnabas, Shelise Y Brooks1, Holly Coleman1, Payam Haghighi1, Shi-Ling Ho1, Karen Schandler1, Sirintorn Stantripop1, Jennifer L Vogt1, Pamela J Thomas1, NISC Comparative Sequencing Program1,2, Gerard G Bouffard1,2, Eric D Green1,2* Abstract Background: The approaches for shotgun-based sequencing of vertebrate genomes are now well-established, and have resulted in the generation of numerous draft whole-genome sequence assemblies. In contrast, the process of refining those assemblies to improve contiguity and increase accuracy (known as ‘sequence finishing’) remains tedious, labor-intensive, and expensive. As a result, the vast majority of vertebrate genome sequences generated to date remain at a draft stage. Results: To date, our genome sequencing efforts have focused on comparative studies of targeted genomic regions, requiring sequence finishing of large blocks of orthologous sequence (average size 0.5-2 Mb) from various subsets of 75 vertebrates. This experience has provided a unique opportunity to compare the relative effort required to finish shotgun-generated genome sequence assemblies from different species, which we report here. Importantly, we found that the sequence assemblies generated for the same orthologous regions from various vertebrates show substantial variation with respect to misassemblies and, in particular, the frequency and characteristics of sequence gaps. As a consequence, the work required to finish different species’ sequences varied greatly. Application of the same standardized methods for finishing provided a novel opportunity to “assay” characteristics of genome sequences among many vertebrate species. It is important to note that many of the problems we have encountered during sequence finishing reflect unique architectural features of a particular vertebrate’s genome, which in some cases may have important functional and/or evolutionary implications. Finally, based on our analyses, we have been able to improve our procedures to overcome some of these problems and to increase the overall efficiency of the sequence-finishing process, although significant challenges still remain. Conclusion: Our findings have important implications for the eventual finishing of the draft whole-genome sequences that have now been generated for a large number of vertebrates. Background Currently, the standard and most efficient approach for the de novo generation of sequence from complex gen- omes (e.g., those of vertebrates) is ‘shotgun sequencing’ [1,2], although new technologies are emerging. In the initial ‘shotgun’ phase, large numbers of overlapping sequence reads are obtained at random from a large- * Correspondence: egreen@nhgri.nih.gov 1NIH Intramural Sequencing Center (NISC), National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA insert clone or a whole genome, yielding highly redun- dant sequence data that typically represent the vast majority of the starting DNA. In the second ‘finishing’ (or refinement) phase, the resulting draft sequence assemblies are analyzed and refined, often with addi- tional sequence data generated to improve continuity and accuracy. Through considerable effort, the sequence can be refined to near perfection–something sought by the Human Genome Project in generating a finished sequence of the human genome [3,4]. However, the gen- eration of such high-quality genomic sequence requires © 2010 Blakesley et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.