Glenn Tesler

SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing

The lions share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

QUAST: quality assessment tool for genome assemblies

SUMMARY Limitations of genome sequencing techniques have led to dozens of assembly algorithms, none of which is perfect. A number of methods for comparing assemblers have been developed, but none is yet a recognized benchmark. Further, most existing methods for comparing assemblies are only applicable to new assemblies of finished genomes; the problem of evaluating assemblies of previously unsequenced species has not been adequately considered. Here, we present QUAST-a quality assessment tool for evaluating and comparing genome assemblies. This tool improves on leading assembly comparison software with new ideas and quality metrics. QUAST can evaluate assemblies both with a reference genome, as well as without a reference. QUAST produces many reports, summary tables and plots to help scientists in their research and in their publications. In this study, we used QUAST to compare several genome assemblers on three datasets. QUAST tables and plots for all of them are available in the Supplementary Material, and interactive versions of these reports are on the QUAST website. AVAILABILITY http://bioinf.spbau.ru/quast . SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Mapping single molecule sequencing reads using basic local alignment with successive refinement (BLASR): application and theory

BackgroundRecent methods have been developed to perform high-throughput sequencing of DNA by Single Molecule Sequencing (SMS). While Next-Generation sequencing methods may produce reads up to several hundred bases long, SMS sequencing produces reads up to tens of kilobases long. Existing alignment methods are either too inefficient for high-throughput datasets, or not sensitive enough to align SMS reads, which have a higher error rate than Next-Generation sequencing.ResultsWe describe the method BLASR (Basic Local Alignment with Successive Refinement) for mapping Single Molecule Sequencing (SMS) reads that are thousands of bases long, with divergence between the read and genome dominated by insertion and deletion error. The method is benchmarked using both simulated reads and reads from a bacterial sequencing project. We also present a combinatorial model of sequencing error that motivates why our approach is effective.ConclusionsThe results indicate that it is possible to map SMS reads with high accuracy and speed. Furthermore, the inferences made on the mapability of SMS reads using our combinatorial model of sequencing error are in agreement with the mapping accuracy demonstrated on simulated reads.

Human and mouse genomic sequences reveal extensive breakpoint reuse in mammalian evolution

The human and mouse genomic sequences provide evidence for a larger number of rearrangements than previously thought and reveal extensive reuse of breakpoints from the same short fragile regions. Breakpoint clustering in regions implicated in cancer and infertility have been reported in previous studies; we report here on breakpoint clustering in chromosome evolution. This clustering reveals limitations of the widely accepted random breakage theory that has remained unchallenged since the mid-1980s. The genome rearrangement analysis of the human and mouse genomes implies the existence of a large number of very short “hidden” synteny blocks that were invisible in the comparative mapping data and ignored in the random breakage model. These blocks are defined by closely located breakpoints and are often hard to detect. Our results suggest a model of chromosome evolution that postulates that mammalian genomes are mosaics of fragile regions with high propensity for rearrangements and solid regions with low propensity for rearrangements.

How to apply de Bruijn graphs to genome assembly

A mathematical concept known as a de Bruijn graph turns the formidable challenge of assembling a contiguous genome from billions of short sequencing reads into a tractable computational problem.The development of algorithmic ideas for Next-Generation Sequencing (NGS) can be traced back three hundred years to the Prussian city of Königsberg (present-day Kaliningrad, Russia), where seven bridges joined the four parts of the city located on opposing banks of the Pregel River and two river islands (Fig. 1a). Königsberg’s residents enjoyed strolling through the city, and they wondered: is it possible to visit every part of the city by walking across each of the seven bridges exactly once and returning to one’s starting location? Remarkably, the conceptual breakthrough used in 1735 to solve this Bridges of Königsberg Problem by the great mathematician Leonhard Euler1 also enables the assembly of billions of short sequencing reads.

GRIMM: genome rearrangements web server

SUMMARY Genome Rearrangements In Man and Mouse (GRIMM) is a tool for analyzing rearrangements of gene orders in pairs of unichromosomal and multichromosomal genomes, with either signed or unsigned gene data. Although there are several programs for analyzing rearrangements in unichromosomal genomes, this is the first to analyze rearrangements in multichromosomal genomes. GRIMM also provides a new algorithm for analyzing comparative maps for which gene directions are unknown. AVAILABILITY A web server, with instructions and sample data, is available at http://www-cse.ucsd.edu/groups/bioinformatics/GRIMM.

Efficient de novo assembly of single-cell bacterial genomes from short-read data sets

Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of DNA from single cells of bacteria that cannot be cultured. Assembling a genome is challenging, however, because MDA generates highly nonuniform coverage of the genome. Here we describe an algorithm tailored for short-read data from single cells that improves assembly through the use of a progressively increasing coverage cutoff. Assembly of reads from single Escherichia coli and Staphylococcus aureus cells captures >91% of genes within contigs, approaching the 95% captured from an assembly based on many E. coli cells. We apply this method to assemble a genome from a single cell of an uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. Our approach enables acquisition of genome assemblies for individual uncultivated bacteria using only short reads, providing cell-specific genetic information absent from metagenomic studies.Whole genome amplification by the multiple displacement amplification (MDA) method allows sequencing of genomes from single cells of bacteria that cannot be cultured. However, genome assembly is challenging because of highly non-uniform read coverage generated by MDA. We describe an improved assembly approach tailored for single cell Illumina sequences that incorporates a progressively increasing coverage cutoff. This allows variable coverage datasets to be utilized effectively with assembly of E. coli and S. aureus single cell reads capturing >91% of genes within contigs, approaching the 95% captured from a multi-cell E. coli assembly. We apply this method to assemble a single cell genome of the uncultivated SAR324 clade of Deltaproteobacteria, a cosmopolitan bacterial lineage in the global ocean. Metabolic reconstruction suggests that SAR324 is aerobic, motile and chemotaxic. These new methods enable acquisition of genome assemblies for individual uncultivated bacteria, providing cell-specific genetic information absent from metagenomic studies.

Efficient algorithms for multichromosomal genome rearrangements

Hannenhalli and Pevzner (36th Annual Symposium on Foundations of Computer Science, Milwaukee, WI, IEEE Computer Soc. Press, Los Alamitos, CA, 1995, p. 581) gave a polynomial time algorithm for computing the minimum number of reversals, translocations, fissions, and fusions, that would transform one multichromosomal genome to another when both have the same set of genes without repeats. We fixed some problems with the construction: (1) They claim it can exhibit such a sequence of steps, but there was a gap in the construction. (2) Their construction had an asymmetry in the number of chromosomes in the two genomes, whereby forward scenarios could have fissions but not fusions.We also improved the speed by combining the algorithm with the algorithm of Bader et al. (J. Comput. Biol. 8 (5) (2001) 483) that computes reversal distances for permutations in linear time.

Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum

Significance This research highlights the discovery and genome reconstruction of a member of the globally distributed yet uncultivated candidate phylum TM6 (designated TM6SC1). In addition to the 16S rRNA gene, no other genomic information is available for this cosmopolitan phylum. This report also introduces a mini-metagenomic approach based on the use of high-throughput single-cell genomics techniques and assembly tools that address a widely recognized issue: how to effectively capture and sequence the currently uncultivated bacterial species that make up the “dark matter of life.” Amplification and sequencing random pools of 100 events enabled an estimated 90% recovery of the TM6SC1 genome. The “dark matter of life” describes microbes and even entire divisions of bacterial phyla that have evaded cultivation and have yet to be sequenced. We present a genome from the globally distributed but elusive candidate phylum TM6 and uncover its metabolic potential. TM6 was detected in a biofilm from a sink drain within a hospital restroom by analyzing cells using a highly automated single-cell genomics platform. We developed an approach for increasing throughput and effectively improving the likelihood of sampling rare events based on forming small random pools of single-flow–sorted cells, amplifying their DNA by multiple displacement amplification and sequencing all cells in the pool, creating a “mini-metagenome.” A recently developed single-cell assembler, SPAdes, in combination with contig binning methods, allowed the reconstruction of genomes from these mini-metagenomes. A total of 1.07 Mb was recovered in seven contigs for this member of TM6 (JCVI TM6SC1), estimated to represent 90% of its genome. High nucleotide identity between a total of three TM6 genome drafts generated from pools that were independently captured, amplified, and assembled provided strong confirmation of a correct genomic sequence. TM6 is likely a Gram-negative organism and possibly a symbiont of an unknown host (nonfree living) in part based on its small genome, low-GC content, and lack of biosynthesis pathways for most amino acids and vitamins. Phylogenomic analysis of conserved single-copy genes confirms that TM6SC1 is a deeply branching phylum.

Matchings in Graphs on Non-orientable Surfaces

We generalize Kasteleyns method of enumerating the perfect matchings in a planar graph to graphs embedding on an arbitrary compact boundaryless 2-manifold S. Kasteleyn stated that perfect matchings in a graph embedding on a surface of genus g could be enumerated as a linear combination of 4g Pfaffians of modified adjacency matrices of the graph. We give an explicit construction that not only does this, but also does it for graphs embedding on non-orientable surfaces. If a graph embeds on the connected sum of a genus g surface with a projective plane (respectively, Klein bottle), the number of perfect matchings can be computed as a linear combination of 22g+1 (respectively, 22g+2) Pfaffians. Thus for any S, this is 22??(S) Pfaffians. We also introduce “crossing orientations,” the analogue of Kasteleyns “admissible orientations” in our context, describing how the Pfaffian of a signed adjacency matrix of a graph gives the sign of each perfect matching according to the number of edge-crossings in the matching. Finally, we count the perfect matchings of an m×n grid on a Mobius strip.