Eugene W. Myers

Basic Local Alignment Search Tool

A new approach to rapid sequence comparison, basic local alignment search tool (BLAST), directly approximates alignments that optimize a measure of local similarity, the maximal segment pair (MSP) score. Recent mathematical results on the stochastic properties of MSP scores allow an analysis of the performance of this method as well as the statistical significance of alignments it generates. The basic algorithm is simple and robust; it can be implemented in a number of ways and applied in a variety of contexts including straightforward DNA and protein sequence database searches, motif searches, gene identification searches, and in the analysis of multiple regions of similarity in long DNA sequences. In addition to its flexibility and tractability to mathematical analysis, BLAST is an order of magnitude faster than existing sequence comparison tools of comparable sensitivity.

Genome sequencing in microfabricated high-density picolitre reactors

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.

Optimal alignments in linear space

Space, not time, is often the limiting factor when computing optimal sequence alignments, and a number of recent papers in the biology literature have proposed space-saving strategies. However, a 1975 computer science paper by Hirschberg presented a method that is superior to the new proposals, both in theory and in practice. The goal of this paper is to give Hirschbergs idea the visibility it deserves by developing a linear-space version of Gotohs algorithm, which accommodates affine gap penalties. A portable C-software package implementing this algorithm is available on the BIONET free of charge.

AnO(ND) difference algorithm and its variations

The problems of finding a longest common subsequence of two sequencesA andB and a shortest edit script for transformingA intoB have long been known to be dual problems. In this paper, they are shown to be equivalent to finding a shortest/longest path in an edit graph. Using this perspective, a simpleO(ND) time and space algorithm is developed whereN is the sum of the lengths ofA andB andD is the size of the minimum edit script forA andB. The algorithm performs well when differences are small (sequences are similar) and is consequently fast in typical applications. The algorithm is shown to haveO(N+D2) expected-time performance under a basic stochastic model. A refinement of the algorithm requires onlyO(N) space, and the use of suffix trees leads to anO(N logN+D2) time variation.

Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing

The spread of multidrug-resistant Staphylococcus aureus (MRSA) strains in the clinical environment has begun to pose serious limits to treatment options. Yet virtually nothing is known about how resistance traits are acquired in vivo. Here, we apply the power of whole-genome sequencing to identify steps in the evolution of multidrug resistance in isogenic S. aureus isolates recovered periodically from the bloodstream of a patient undergoing chemotherapy with vancomycin and other antibiotics. After extensive therapy, the bacterium developed resistance, and treatment failed. Sequencing the first vancomycin susceptible isolate and the last vancomycin nonsusceptible isolate identified genome wide only 35 point mutations in 31 loci. These mutations appeared in a sequential order in isolates that were recovered at intermittent times during chemotherapy in parallel with increasing levels of resistance. The vancomycin nonsusceptible isolates also showed a 100-fold decrease in susceptibility to daptomycin, although this antibiotic was not used in the therapy. One of the mutated loci associated with decreasing vancomycin susceptibility (the vraR operon) was found to also carry mutations in six additional vancomycin nonsusceptible S. aureus isolates belonging to different genetic backgrounds and recovered from different geographic sites. As costs drop, whole-genome sequencing will become a useful tool in elucidating complex pathways of in vivo evolution in bacterial pathogens.

V3D enables real-time 3D visualization and quantitative analysis of large-scale biological image data sets

The V3D system provides three-dimensional (3D) visualization of gigabyte-sized microscopy image stacks in real time on current laptops and desktops. V3D streamlines the online analysis, measurement and proofreading of complicated image patterns by combining ergonomic functions for selecting a location in an image directly in 3D space and for displaying biological measurements, such as from fluorescent probes, using the overlaid surface objects. V3D runs on all major computer platforms and can be enhanced by software plug-ins to address specific biological problems. To demonstrate this extensibility, we built a V3D-based application, V3D-Neuron, to reconstruct complex 3D neuronal structures from high-resolution brain images. V3D-Neuron can precisely digitize the morphology of a single neuron in a fruitfly brain in minutes, with about a 17-fold improvement in reliability and tenfold savings in time compared with other neuron reconstruction tools. Using V3D-Neuron, we demonstrate the feasibility of building a 3D digital atlas of neurite tracts in the fruitfly brain.

Finishing a whole-genome shotgun: Release 3 of the Drosophila melanogaster euchromatic genome sequence

BackgroundThe Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.ResultsOur finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp.ConclusionsThe WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis.

PILER: identification and classification of genomic repeats

SUMMARY Repeated elements such as satellites and transposons are ubiquitous in eukaryotic genomes. De novo computational identification and classification of such elements is a challenging problem. Therefore, repeat annotation of sequenced genomes has historically largely relied on sequence similarity to hand-curated libraries of known repeat families. We present a new approach to de novo repeat annotation that exploits characteristic patterns of local alignments induced by certain classes of repeats. We describe PILER, a package of efficient search algorithms for identifying such patterns. Novel repeats found using PILER are reported for Homo sapiens, Arabidopsis thalania and Drosophila melanogaster. AVAILABILITY The PILER software is freely available at http://www.drive5.com/piler.

The fragment assembly string graph

We present a concept and formalism, the string graph, which represents all that is inferable about a DNA sequence from a collection of shotgun sequencing reads collected from it. We give time and space efficient algorithms for constructing a string graph given the collection of overlaps between the reads and, in particular, present a novel linear expected time algorithm for transitive reduction in this context. The result demonstrates that the decomposition of reads into kmers employed in the de Bruijn graph approach described earlier is not essential, and exposes its close connection to the unitig approach we developed at Celera. This paper is a preliminary piece giving the basic algorithm and results that demonstrate the efficiency and scalability of the method. These ideas are being used to build a next-generation whole genome assembler called BOA (Berkeley Open Assembler) that will easily scale to mammalian genomes.

Toward simplifying and accurately formulating fragment assembly

The fragment assembly problem is that of reconstructing a DNA sequence from a collection of randomly sampled fragments. Traditionally, the objective of this problem has been to produce the shortest string that contains all the fragments as substrings, but in the case of repetitive target sequences this objective produces answers that are overcompressed. In this paper, the problem is reformulated as one of finding a maximum-likelihood reconstruction with respect to the two-sided Kolmogorov-Smirnov statistic, and it is argued that this is a better formulation of the problem. Next the fragment assembly problem is recast in graph-theoretic terms as one of finding a noncyclic subgraph with certain properties and the objectives of being shortest or maximally likely are also recast in this framework. Finally, a series of graph reduction transformations are given that dramatically reduce the size of the graph to be explored in practical instances of the problem. This reduction is very important as the underlying problems are NP-hard. In practice, the transformed problems are so small that simple branch-and-bound algorithms successfully solve them, thus permitting auxiliary experimental information to be taken into account in the form of overlap, orientation, and distance constraints.