Blakesley et al. BMC Genomics 2010, 11:21 http://www.biomedcentral.com/1471-2164/11/21 Analyzer) can recover some of the sequence that is otherwise missing in initial assemblies. We are investi- gating further alternative approaches for employing these new platforms in generating sequence data that are more complete, reducing the effort required for fin- ishing (unpublished data). However, our experience also confirms the continued labor-intensive nature of sequence finishing and the absence of any ‘magic bullet’ that resolves all major sequence-finishing problems. Some of the most challen- ging sequences to finish are from species for which a draft genome sequence has already been generated and currently sits at a draft stage, such as for dog [37] and platypus [38]. In fact, draft whole-genome sequences of various levels of completion have been generated for most of the more than 30 species studied here [8,9]. Our findings may provide important insights that will aid future efforts to refine those draft sequence assem- blies. If high-quality finished sequences are eventually to be produced for all of these vertebrate genomes, then continued vigilance about understanding the general and species-specific challenges associated with sequence finishing will be needed, as will methodological advances to further reduce the cost and to improve the efficiency of the sequence-finishing process. Methods Sequence generation Purified BAC DNA was sheared and ‘shotgun subcloned’ into plasmids containing 3- to 5- kb inserts using E. coli DH10B tonA host cells (Invitrogen). Plasmid DNA was purified from individual subclones by an automated magnetic bead process (Agencourt Bioscience). Paired sequence reads were generated from randomly selected subclones by universal forward- and reverse-primed fluorescent dideoxy sequencing reactions [1,2] using AB3730xl sequencing instruments (Applied Biosystems). Typically, 2,500-2,800 sequence reads were generated for each BAC (which averaged ~ 165 kb in size), with the resulting Phrap-based [25,26] sequence assembly consistently providing at least eight-fold redundancy of the starting BAC. Sequence finishing Sequence assemblies were refined to produce compara- tive-grade finished sequence; this process, described in detail by Blakesley et al. [5], including a summary of the step-by-step procedures within the Supplementary Materials of that paper, mostly involved a series of com- putational procedures that yields an ordered and oriented map of all sequence contigs greater than 2 kb in size. That map was routinely verified with indepen- dent data, including sequence derived from overlapping BACs, alignments with orthologous sequence from another species, and/or results of PCR-based Page 13 of 15 experiments to confirm contig adjacency. A subset of the BACs was further finished to the ‘human-grade’ standards established for sequencing the human genome [4,39]. The GenBank accession numbers for all sequenced BACs analyzed here are available on request. All sequencing reactions for the shotgun phase and most reactions for the finishing phase employed Big-Dye Terminator chemistry (Applied Biosystems) as described by the manufacturer. Certain other sequence finishing reactions used alternative chemistries, where dGTP Big- Dye Terminator Mix (Applied Biosystems) or an 80:20 mixture of BigDye:dGTP Big Dye substituted for BigDye Terminator Mix in the sequencing reaction mixture. In certain cases, one-tenth volume of SeqRxA (Invitrogen) was added to the reaction. Acknowledgements We thank all participants of the NISC Comparative Sequencing Program (participants include A Antonellis, K Ayele, D Bass, SM Beckstrom-Sternberg, CP Brinkley, K Cariaga, G Chu, I Chub, M Dekhtyar, NL Dietrich, J Engle, T Fuksenko, M Gestole, S Granite, M Gregory, X Guan, N Gurson, P Hall, E Han, J Han, A Hargrove, K Hines-Harris, J Hodax, P Hu, B Hurle, JR Idol, T Johnson, E Karlins, P Laric, SQ Lee-Lin, R Legaspi, S Lovett, M Madden, QL Maduro, VB Maduro, EH Margulies, CA Masiello, SD Mastrian, C Montemayor, JC Mullikin, J Myrick, E Novotny, M Park, R Pearson, A Palumbo, ME Portnoy, AB Prasad, O Puri, C Ramsahoye, N Reddix-Dugue, N Riebow, A Sante, B Schmidt, MG Schueler, C Sison, L Smith, A Taye, JW Thomas, EE Tiongson, JW Touchman, C Tsurgeon, V Tsipouri, MA Walker, KD Wetherby, LS Wiggins, TR Withers, and AC Young) for their contributions to the studies described here. We also thank Hatice Abaan, Paul Havlak, and Elliott Margulies (NHGRI) for sharing unpublished Genome Analyzer sequencing data. We thank Kim Kyung and Robert Fulton (WUGSC) for providing sequence of the small gap in our dog BAC [GenBank: AC090445]. We also thank Alice Young (NISC), Rick Myers, and Richards Gibbs for critical feedback about this manuscript. This work was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health. Author details 1NIH Intramural Sequencing Center (NISC), National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2Genome Technology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA. Authors’ contributions RWB, NFH, JCM, BM, GGB, and EDG conceived the study. NFH led the bioinformatics analyses, with contributions from JCM, BM, GGB, and RWB. JG, BBB, SYB, HC, PH, S-LH, KS, SS, JLV, PJT and members of the NISC Comparative Sequencing Program generated the data and performed some of the bioinformatic analyses. RWB, NFH, and EDG wrote the manuscript. Received: 11 March 2009 Accepted: 11 January 2010 Published: 11 January 2010 References 1. Wilson RK, Mardis ER: Shotgun sequencing. Genome Analysis: A laboratory manual: Analyzing DNA Cold Spring Harbor, NY: Cold Spring Harbor Laboratory PressBirren B, Green ED, Klapholz S, Myers RM, Roskams J 1997, 1:397-454. 2. Green ED: Strategies for the systematic sequencing of complex genomes. Nat Rev Genet 2001, 2:573-583. 3. International Human Genome Sequencing Consortium: Initial sequencing and analysis of the human genome. Nature 2001, 409:860-921. 4. International Human Genome Sequencing Consortium: Finishing the euchromatic sequence of the human genome. Nature 2004, 431:931-945.