2× genomes—Does depth matter?

This issue of Genome Research marks the publication by collaborators at the NIH, Agencourt Bioscience, and the Broad Institute of a genome sequence for the domestic cat Felis catus (Pontius et al. 2007). It was obtained by a whole-genome shotgun approach in which, on average, each genomic base is represented in roughly two sequence reads (“2 ” redundancy), a level at which there remain many gaps in the sequence due to statistical fluctuations in read placement, biases in subclone libraries, and assembly difficulties. In all, the NHGRI is sponsoring 24 such “low redundancy” mammalian genome sequences, 17 of which have already been assembled and released (Table 1). Complete coverage of every genome would obviously be preferable; the decision to acquire multiple incomplete sequences represents a compromise balancing phylogenetic breadth (inclusion of many species) against redundancy depth for particular species. In this commentary, I discuss some of the issues involved in this compromise, and touch on the uses, characteristics, and limitations of 2 assemblies. The tradeoff between breadth and depth has actually been a recurring theme in genome sequencing, because the choice between spreading available data-generating capacity broadly over a larger extent of targeted DNA and obtaining deeper coverage of a more limited target arises in several contexts. The appropriate balance in each case is far from obvious and depends on a number of factors, including the relative difficulty of obtaining DNA sources of various types, available data analysis tools, and the implications of sequence errors and gaps for downstream utility. Early in the Human Genome Project, the high level of redundancy required by the shotgun method was often viewed as unduly wasteful, and considerable effort was expended toward developing more efficient directed strategies. These all foundered, due to a combination of logistical complexity, the problems posed by interspersed repeats, and the recognition that variation in data quality makes it impossible to get highly accurate sequence without considerable redundancy. Once the superiority of the shotgun approach was accepted, a key issue became the breadth of the targeted region: is it better to shotgun a whole genome, or a series of individual BAC clones (Green 1997; Weber and Myers 1997)? Although not immediately obvious, this choice also involves a breadth versus depth tradeoff, in two ways: with whole genome targets, typically 25% of reads cannot be assembled (Adams et al. 2000; Venter et al. 2001), thereby reducing the overall effective depth, and, in addition, the inability to control redundancy depth in local regions (as can readily be done in a clone-by-clone approach) implies that a larger fraction of the genome will be poorly covered. The whole-genome approach is also unable to resolve many segmental duplications (She et al. 2004) and interspersed repeats (Adams et al. 2000). Nonetheless, the (considerable) advantage of simpler logistics, combined with a willingness to tolerate less accurate and less complete sequence, have made the whole-genome strategy the current method of choice for sequencing most genomes. With completion of the human genome sequence, the breadth versus depth issue has now shifted to how best to apply available sequencing capacity across organisms. Broad phylogenetic representation accelerates research on a wide variety of species and provides general insights into the core evolutionary processes of mutation and selection. However, from the perspective of the human genome, its main benefit is to help identify functional features as regions with a reduced frequency of substitution differences between organisms due to purifying selection. The statistical power to detect such regions depends on overall sequence divergence: there is an analogy to shotgunsequence assembly, in that one seeks “coverage” of human bases by multiply aligned orthologous sequences instead of reads, and the relevant “depth” is total branch length (expected number of mutations per neutral site) of the phylogenetic tree relating the species. Theoretical analysis (Eddy 2005) indicates that a surprisingly large branch length is required to resolve small features with reasonable power—for example, the mouse genome, which among placental mammals is one of the most highly diverged from human, yields only enough information to identify conserved features longer than 50 bp. While there is substantial branch length available from sequenced non-mammalian vertebrates, they provide no information regarding mammal-specific features. The 24 NHGRI 2 targets, combined with the seven or so other placental mammals (human, mouse, rat, dog, chimpanzee, macaque, cow) having more complete sequences, are predicted to provide adequate branch length to resolve features of the size of a typical protein-binding site (6–8 bp) at a false positive rate of one per 10 kb (http://www.genome.gov/25521745). This resolution could not be achieved by allocating the same number of sequence reads to six genomes at a more conventional 8 redundancy, or even 12 genomes at 4 . It is worth noting that effective delineation of conserved elements via this strategy is not yet a completely solved problem. Multiple-genome alignments are error prone even with relatively complete sequences (Prakash and Tompa 2007). Perhaps more seriously, comprehensive analysis of local genomic regions (ENCODE Project Consortium 2007) suggests that, at least with current methods, sequence conservation fails to detect some important features, even when alignments appear reliable. This likely reflects, at least in part, the intrinsic plasticity of the sequence signals involved, and it remains unclear to what extent improvements in computational strategies or additional sequence data will be able to address this. What are the consequences of reduced depth for individual genomes? The primary determinants of sequence utility are assembly accuracy (correctness of read overlaps and of contig order and orientation) and percent coverage of the genome. For lowredundancy (∼0.5 to ∼3 ) shotguns, both accuracy and coverage can be improved by ‘assisting’ the assembly using available near-complete “reference” genomes from other species. Cat is a E-mail phg@u.washington.edu; fax (206) 685-9720. Article is online at http://www.genome.org/cgi/doi/10.1101/gr.7050807. Freely available online through the Genome Research Open Access option. Commentary