Search

Exceptionally accurate genome reference sequences have proven to be of great value to microbial researchers. Thus, to date, about 1800 bacterial genome assemblies have been "finished" at great expense with the aid of manual laboratory and computational processes that typically iterate over a period of months or even years. By applying a new laboratory design and new assembly algorithm to 16 samples, we demonstrate that assemblies exceeding finished quality can be obtained from whole-genome shotgun data and automated computation. Cost and time requirements are thus dramatically reduced.

Eliminating the bacterial cloning step has been a major factor in the vastly improved efficiency of massively parallel sequencing approaches. However, this also has made it a technical challenge to produce the modern equivalent of the Fosmid- or BAC-end sequences that were crucial for assembling and analyzing complex genomes during the Sanger-based sequencing era. To close this technology gap, we developed Fosill, a method for converting Fosmids to Illumina-compatible jumping libraries. We constructed Fosmid libraries in vectors with Illumina primer sequences and specific nicking sites flanking the cloning site. Our family of pFosill vectors allows multiplex Fosmid cloning of end-tagged genomic fragments without physical size selection and is compatible with standard and multiplex paired-end Illumina sequencing. To excise the bulk of each cloned insert, we introduced two nicks in the vector, translated them into the inserts, and cleaved them. Recircularization of the vector via coligation of insert termini followed by inverse PCR generates a jumping library for paired-end sequencing with 101-base reads. The yield of unique Fosmid-sized jumps is sufficiently high, and the background of short, incorrectly spaced and chimeric artifacts sufficiently low, to enable applications such as mapping of structural variation and scaffolding of de novo assemblies. We demonstrate the power of Fosill to map genome rearrangements in a cancer cell line and identified three fusion genes that were corroborated by RNA-seq data. Our Fosill-powered assembly of the mouse genome has an N50 scaffold length of 17.0 Mb, rivaling the connectivity (16.9 Mb) of the Sanger-sequencing based draft assembly.

New DNA sequencing technologies deliver data at dramatically lower costs but demand new analytical methods to take full advantage of the very short reads that they produce. We provide an initial, theoretical solution to the challenge of de novo assembly from whole-genome shotgun "microreads." For 11 genomes of sizes up to 39 Mb, we generated high-quality assemblies from 80x coverage by paired 30-base simulated reads modeled after real Illumina-Solexa reads. The bacterial genomes of Campylobacter jejuni and Escherichia coli assemble optimally, yielding single perfect contigs, and larger genomes yield assemblies that are highly connected and accurate. Assemblies are presented in a graph form that retains intrinsic ambiguities such as those arising from polymorphism, thereby providing information that has been absent from previous genome assemblies. For both C. jejuni and E. coli, this assembly graph is a single edge encompassing the entire genome. Larger genomes produce more complicated graphs, but the vast majority of the bases in their assemblies are present in long edges that are nearly always perfect. We describe a general method for genome assembly that can be applied to all types of DNA sequence data, not only short read data, but also conventional sequence reads.