Assembly of large genomes using second-generation sequencing

Second-generation sequencing technology can now be used to sequence an entire human genome in a matter of days and at low cost. Sequence read lengths, initially very short, have rapidly increased since the technology first appeared, and we now are seeing a growing number of efforts to sequence large genomes de novo from these short reads. In this Perspective, we describe the issues associated with short-read assembly, the different types of data produced by second-gen sequencers, and the latest assembly algorithms designed for these data. We also review the genomes that have been assembled recently from short reads and make recommendations for sequencing strategies that will yield a high-quality assembly.As genome sequencing technology has evolved, methods for assembling genomes have changed with it. Genome sequencers have never been able to “read” more than a relatively short stretch of DNA at once, with read lengths gradually increasing over time. Reconstructing a complete genome from a set of reads requires an assembly program, and a variety of genome assemblers have been used for this task. In 1995, when the first bacterial genome was published (Haemophilus influenzae), read lengths were ∼460 base pairs (bp), and that whole-genome shotgun (WGS) sequencing project generated 24,304 reads (Fleischmann et al. 1995). The human genome project required ∼30 million reads, with lengths up to 800 bp, using Sanger sequencing technology and automated capillary sequencers (International Human Genome Sequencing Consortium 2001; Venter et al. 2001). This corresponded to 24 billion bases (Gb), or approximately eightfold coverage of the 3-Gb human genome. Redundant coverage, in which on average every nucleotide is sequenced many times over, is required to produce a high-quality assembly. Another benefit of redundancy is greatly increased accuracy compared with a single read: Where a single read might have an error rate of 1%, eightfold coverage has an error rate as low as 10⁻¹⁶ when eight high-quality reads agree with one another. High coverage is also necessary to sequence polymorphic alleles within diploid or polyploid genomes.Current second-generation sequencing (SGS) technologies produce read lengths ranging from 35 to 400 bp, at far greater speed and much lower cost than Sanger sequencing. However, as reads get shorter, coverage needs to increase to compensate for the decreased connectivity and produce a comparable assembly. Certain problems cannot be overcome by deeper coverage: If a repetitive sequence is longer than a read, then coverage alone will never compensate, and all copies of that sequence will produce gaps in the assembly. These gaps can be spanned by paired reads—consisting of two reads generated from a single fragment of DNA and separated by a known distance—as long as the pair separation distance is longer than the repeat. Paired-end sequencing is available from most of the SGS machines, although it is not yet as flexible or as reliable as paired-end sequencing using traditional methods.After the successful assembly of the human (International Human Genome Sequencing Consortium 2001; Venter et al. 2001) and mouse (Waterston et al. 2002) genomes by whole-genome shotgun sequencing, most large-scale genome projects quickly moved to adopt the WGS approach, which has subsequently been used for dozens of eukaryotic genomes. Today, thanks to changes in sequencing technology, a major question confronting genome projects is, can we sequence a large genome (>100 Mbp) using short reads? If so, what are the limitations on read length, coverage, and error rates? How much paired-end sequencing is necessary? And what will the assembly look like? In this perspective we take a look at each of these questions and describe the solutions available today. Although we provide some answers, we have no doubt that the solutions will change rapidly over the next few years, as both the sequencing methods and the computational solutions improve.