Whole Genome Shotgun Sequencing Data Research Articles

Organellar Genomes of White Spruce (Picea glauca): Assembly and Annotation.

The genome sequences of the plastid and mitochondrion of white spruce (Picea glauca) were assembled from whole-genome shotgun sequencing data using ABySS. The sequencing data contained reads from both the nuclear and organellar genomes, and reads of the organellar genomes were abundant in the data as each cell harbors hundreds of mitochondria and plastids. Hence, assembly of the 123-kb plastid and 5.9-Mb mitochondrial genomes were accomplished by analyzing data sets primarily representing low coverage of the nuclear genome. The assembled organellar genomes were annotated for their coding genes, ribosomal RNA, and transfer RNA. Transcript abundances of the mitochondrial genes were quantified in three developmental tissues and five mature tissues using data from RNA-seq experiments. C-to-U RNA editing was observed in the majority of mitochondrial genes, and in four genes, editing events were noted to modify ACG codons to create cryptic AUG start codons. The informatics methodology presented in this study should prove useful to assemble organellar genomes of other plant species using whole-genome shotgun sequencing data.

Genomic analysis of a migratory divide reveals candidate genes for migration and implicates selective sweeps in generating islands of differentiation.

Differential gene flow, reductions in diversity following linked selection and/or features of the genome can structure patterns of genomic differentiation during the process of speciation. Possible sources of reproductive isolation are well studied between coastal and inland subspecies groups of Swainson's thrushes, with differences in seasonal migratory behaviour likely playing a key role in reducing hybrid fitness. We assembled and annotated a draft reference genome for this species and generated whole-genome shotgun sequence data for populations adjacent to the hybrid zone between these groups. We documented substantial genomewide heterogeneity in relative estimates of genetic differentiation between the groups. Within population diversity was lower in areas of high relative differentiation, supporting a role for selective sweeps in generating this pattern. Absolute genetic differentiation was reduced in these areas, further suggesting that recurrent selective sweeps in the ancestral population and/or between divergent populations following secondary contact likely occurred. Relative genetic differentiation was also higher near centromeres and on the Z chromosome, suggesting that features of the genome also contribute to genomewide heterogeneity. Genes linked to migratory traits were concentrated in islands of differentiation, supporting previous suggestions that seasonal migration is under divergent selection between Swainson's thrushes. Differences in migratory behaviour likely play a central role in the speciation of many taxa; we developed the infrastructure here to permit future investigations into the role several candidate genes play in reducing gene flow between not only Swainson's thrushes but other species as well.

Genetic anchoring of whole-genome shotgun assemblies.

The recent advances in sequencing throughput and genome assembly algorithms have established whole-genome shotgun (WGS) assemblies as the cornerstone of the genomic infrastructure for many species. WGS assemblies can be constructed with comparative ease and give a comprehensive representation of the gene space even of large and complex genomes. One major obstacle in utilizing WGS assemblies for important research applications such as gene isolation or comparative genomics has been the lack of chromosomal positioning and contextualization of short sequence contigs. Assigning chromosomal locations to sequence contigs required the construction and integration of genome-wide physical maps and dense genetic linkage maps as well as synteny to model species. Recently, methods to rapidly construct ultra-dense linkage maps encompassing millions of genetic markers from WGS sequencing data of segregating populations have made possible the direct assignment of genetic positions to short sequence contigs. Here, we review recent developments in the integration of WGS assemblies and sequence-based linkage maps, discuss challenges for further improvement of the methodology and outline possible applications building on genetically anchored WGS assemblies.

Efficient de novo assembly of large and complex genomes by massively parallel sequencing of Fosmid pools.

BackgroundSampling genomes with Fosmid vectors and sequencing of pooled Fosmid libraries on the Illumina platform for massive parallel sequencing is a novel and promising approach to optimizing the trade-off between sequencing costs and assembly quality.ResultsIn order to sequence the genome of Norway spruce, which is of great size and complexity, we developed and applied a new technology based on the massive production, sequencing, and assembly of Fosmid pools (FP). The spruce chromosomes were sampled with ~40,000 bp Fosmid inserts to obtain around two-fold genome coverage, in parallel with traditional whole genome shotgun sequencing (WGS) of haploid and diploid genomes. Compared to the WGS results, the contiguity and quality of the FP assemblies were high, and they allowed us to fill WGS gaps resulting from repeats, low coverage, and allelic differences. The FP contig sets were further merged with WGS data using a novel software package GAM-NGS.ConclusionsBy exploiting FP technology, the first published assembly of a conifer genome was sequenced entirely with massively parallel sequencing. Here we provide a comprehensive report on the different features of the approach and the optimization of the process.We have made public the input data (FASTQ format) for the set of pools used in this study:ftp://congenie.org/congenie/Nystedt_2013/Assembly/ProcessedData/FosmidPools/.(alternatively accessible via http://congenie.org/downloads).The software used for running the assembly process is available at http://research.scilifelab.se/andrej_alexeyenko/downloads/fpools/.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-439) contains supplementary material, which is available to authorized users.

Use of Whole Genome Shotgun Metagenomics: A Practical Guide for the Microbiome-Minded Physician Scientist

Whole genome shotgun sequencing (WGS) has been increasingly recognized as the most comprehensive and robust approach for metagenomics research. When compared with 16S-based metagenomics, it offers the advantage of identification of species level taxonomy and the estimation of metabolic pathway activities from human and environmental samples. Several large-scale metagenomic projects have been recently conducted or are currently underway utilizing WGS. With the generation of vast amounts of data, the bioinformatics and computational analysis of WGS results become vital for the success of a metagenomics study. However, each step in the WGS data analysis, including metagenome assembly, gene prediction, taxonomy identification, function annotation, and pathway analysis, is complicated by the shear amount of data. Algorithms and tools have been developed specifically to handle WGS-generated metagenomics data with the hope of reducing the requirement on computational time and storage space. Here, we present an overview of the current state of metagenomics through WGS sequencing, challenges frequently encountered, and up-to-date solutions. Several applications that are uniquely applicable to microbiome studies in reproductive and perinatal medicine are also discussed.

A Sequence-Ready Physical Map of Barley Anchored Genetically by Two Million Single-Nucleotide Polymorphisms

Barley (Hordeum vulgare) is an important cereal crop and a model species for Triticeae genomics. To lay the foundation for hierarchical map-based sequencing, a genome-wide physical map of its large and complex 5.1 billion-bp genome was constructed by high-information content fingerprinting of almost 600,000 bacterial artificial chromosomes representing 14-fold haploid genome coverage. The resultant physical map comprises 9,265 contigs with a cumulative size of 4.9 Gb representing 96% of the physical length of the barley genome. The reliability of the map was verified through extensive genetic marker information and the analysis of topological networks of clone overlaps. A minimum tiling path of 66,772 minimally overlapping clones was defined that will serve as a template for hierarchical clone-by-clone map-based shotgun sequencing. We integrated whole-genome shotgun sequence data from the individuals of two mapping populations with published bacterial artificial chromosome survey sequence information to genetically anchor the physical map. This novel approach in combination with the comprehensive whole-genome shotgun sequence data sets allowed us to independently validate and improve a previously reported physical and genetic framework. The resources developed in this study will underpin fine-mapping and cloning of agronomically important genes and the assembly of a draft genome sequence.

The ‘Neolithic’ in Borneo: a different model

The transition from hunting and gathering to farming in Europe was brought upon by arrival of new people carrying novel material culture and genetic ancestry. The exact nature and scale of the transition—both material and genetic—varied in different parts of Europe [1, 2, 3, 4, 5, 6, 7]. Farming-based economies appear relatively late in Northeast Europe, and the extent to which they involve change in genetic ancestry is not fully understood due to the lack of relevant ancient DNA data. Here we present the results from new low-coverage whole-genome shotgun sequence data from five hunter-gatherers and five first farmers of Estonia whose remains date to 4,500 to 6,300 years before present. We find evidence of significant differences between the two groups in the composition of autosomal as well as mtDNA, X chromosome, and Y chromosome ancestries. We find that Estonian hunter-gatherers of Comb Ceramic culture are closest to Eastern hunter-gatherers, which is in contrast to earlier hunter-gatherers from the Baltics, who are close to Western hunter-gatherers [8, 9]. The Estonian first farmers of Corded Ware culture show high similarity in their autosomes with European hunter-gatherers, Steppe Eneolithic and Bronze Age populations, and European Late Neolithic/Bronze Age populations, while their X chromosomes are in addition equally closely related to European and Anatolian and Levantine early farmers. These findings suggest that the shift to intensive cultivation and animal husbandry in Estonia was triggered by the arrival of new people with predominantly Steppe ancestry but whose ancestors had undergone sex-specific admixture with early farmers with Anatolian ancestry.

High throughput sequencing methods for microbiome profiling: application to food animal systems

Analysis of microbial communities using high throughput sequencing methods began in the mid 2000s permitting the production of 1000s to 10,000s of sequence reads per sample and megabases of data per sequence run. This then unprecedented depth of sequencing allowed, for the first time, the discovery of the 'rare biosphere' in environmental samples. The technology was quickly applied to studies in several human subjects. Perhaps these early studies served as a reminder that though the microbes that inhabit mammals are known to outnumber host cells by an order of magnitude or more, most of these are unknown members of our second genome, or microbiome (as coined by Joshua Lederberg), because of our inability to culture them. High throughput methods for microbial 16S ribosomal RNA gene and whole genome shotgun (WGS) sequencing have now begun to reveal the composition and identity of archaeal, bacterial and viral communities at many sites, in and on the human body. Surveys of the microbiota of food production animals have been published in the past few years and future studies should benefit from protocols and tools developed from large-scale human microbiome studies. Nevertheless, production animal-related resources, such as improved host genome assemblies and increased numbers and diversity of host-specific microbial reference genome sequences, will be needed to permit meaningful and robust analysis of 16S rDNA and WGS sequence data.

Exploring single-sample SNP and INDEL calling with whole-genomede novoassembly

Eugene Myers in his string graph paper suggested that in a string graph or equivalently a unitig graph, any path spells a valid assembly. As a string/unitig graph also encodes every valid assembly of reads, such a graph, provided that it can be constructed correctly, is in fact a lossless representation of reads. In principle, every analysis based on whole-genome shotgun sequencing (WGS) data, such as SNP and insertion/deletion (INDEL) calling, can also be achieved with unitigs. To explore the feasibility of using de novo assembly in the context of resequencing, we developed a de novo assembler, fermi, that assembles Illumina short reads into unitigs while preserving most of information of the input reads. SNPs and INDELs can be called by mapping the unitigs against a reference genome. By applying the method on 35-fold human resequencing data, we showed that in comparison to the standard pipeline, our approach yields similar accuracy for SNP calling and better results for INDEL calling. It has higher sensitivity than other de novo assembly based methods for variant calling. Our work suggests that variant calling with de novo assembly can be a beneficial complement to the standard variant calling pipeline for whole-genome resequencing. In the methodological aspects, we propose FMD-index for forward-backward extension of DNA sequences, a fast algorithm for finding all super-maximal exact matches and one-pass construction of unitigs from an FMD-index. http://github.com/lh3/fermi

Comrad: detection of expressed rearrangements by integrated analysis of RNA-Seq and low coverage genome sequence data

Comrad is a novel algorithmic framework for the integrated analysis of RNA-Seq and whole genome shotgun sequencing (WGSS) data for the purposes of discovering genomic rearrangements and aberrant transcripts. The Comrad framework leverages the advantages of both RNA-Seq and WGSS data, providing accurate classification of rearrangements as expressed or not expressed and accurate classification of the genomic or non-genomic origin of aberrant transcripts. A major benefit of Comrad is its ability to accurately identify aberrant transcripts and associated rearrangements using low coverage genome data. As a result, a Comrad analysis can be performed at a cost comparable to that of two RNA-Seq experiments, significantly lower than an analysis requiring high coverage genome data. We have applied Comrad to the discovery of gene fusions and read-throughs in prostate cancer cell line C4-2, a derivative of the LNCaP cell line with androgen-independent characteristics. As a proof of concept, we have rediscovered in the C4-2 data 4 of the 6 fusions previously identified in LNCaP. We also identified six novel fusion transcripts and associated genomic breakpoints, and verified their existence in LNCaP, suggesting that Comrad may be more sensitive than previous methods that have been applied to fusion discovery in LNCaP. We show that many of the gene fusions discovered using Comrad would be difficult to identify using currently available techniques. A C++ and Perl implementation of the method demonstrated in this article is available at http://compbio.cs.sfu.ca/.

Genomic signatures of strain selection and enhancement in Bacillus atrophaeus var. globigii, a historical biowarfare simulant.

BackgroundDespite the decades-long use of Bacillus atrophaeus var. globigii (BG) as a simulant for biological warfare (BW) agents, knowledge of its genome composition is limited. Furthermore, the ability to differentiate signatures of deliberate adaptation and selection from natural variation is lacking for most bacterial agents. We characterized a lineage of BGwith a long history of use as a simulant for BW operations, focusing on classical bacteriological markers, metabolic profiling and whole-genome shotgun sequencing (WGS).ResultsArchival strains and two “present day” type strains were compared to simulant strains on different laboratory media. Several of the samples produced multiple colony morphotypes that differed from that of an archival isolate. To trace the microevolutionary history of these isolates, we obtained WGS data for several archival and present-day strains and morphotypes. Bacillus-wide phylogenetic analysis identified B. subtilis as the nearest neighbor to B. atrophaeus. The genome of B. atrophaeus is, on average, 86% identical to B. subtilis on the nucleotide level. WGS of variants revealed that several strains were mixed but highly related populations and uncovered a progressive accumulation of mutations among the “military” isolates. Metabolic profiling and microscopic examination of bacterial cultures revealed enhanced growth of “military” isolates on lactate-containing media, and showed that the “military” strains exhibited a hypersporulating phenotype.ConclusionsOur analysis revealed the genomic and phenotypic signatures of strain adaptation and deliberate selection for traits that were desirable in a simulant organism. Together, these results demonstrate the power of whole-genome and modern systems-level approaches to characterize microbial lineages to develop and validate forensic markers for strain discrimination and reveal signatures of deliberate adaptation.

Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing

Massively parallel DNA sequencing technologies have greatly increased our ability to generate large amounts of sequencing data at a rapid pace. Several methods have been developed to enrich for genomic regions of interest for targeted sequencing. We have compared three of these methods: Molecular Inversion Probes (MIP), Solution Hybrid Selection (SHS), and Microarray-based Genomic Selection (MGS). Using HapMap DNA samples, we compared each of these methods with respect to their ability to capture an identical set of exons and evolutionarily conserved regions associated with 528 genes (2.61 Mb). For sequence analysis, we developed and used a novel Bayesian genotype-assigning algorithm, Most Probable Genotype (MPG). All three capture methods were effective, but sensitivities (percentage of targeted bases associated with high-quality genotypes) varied for an equivalent amount of pass-filtered sequence: for example, 70% (MIP), 84% (SHS), and 91% (MGS) for 400 Mb. In contrast, all methods yielded similar accuracies of >99.84% when compared to Infinium 1M SNP BeadChip-derived genotypes and >99.998% when compared to 30-fold coverage whole-genome shotgun sequencing data. We also observed a low false-positive rate with all three methods; of the heterozygous positions identified by each of the capture methods, >99.57% agreed with 1M SNP BeadChip, and >98.840% agreed with the whole-genome shotgun data. In addition, we successfully piloted the genomic enrichment of a set of 12 pooled samples via the MGS method using molecular bar codes. We find that these three genomic enrichment methods are highly accurate and practical, with sensitivities comparable to that of 30-fold coverage whole-genome shotgun data.

Comparative Metagenomic Analysis of a Microbial Community Residing at a Depth of 4,000 Meters at Station ALOHA in the North Pacific Subtropical Gyre

The deep sea (water depth of >2,000 m) represents the largest biome on Earth. Yet relatively little is known about its microbial community's structure, function, and adaptation to the cold and deep biosphere. To provide further genomic insights into deep-sea planktonic microbes, we sequenced a total of approximately 200 Mbp of a random whole-genome shotgun (WGS) library from a microbial community residing at a depth of 4,000 m at Station ALOHA in the Pacific Ocean and compared it to other available WGS sequence data from surface and deep waters. Our analyses indicated that the deep-sea lifestyle is likely facilitated by a collection of very subtle adaptations, as opposed to dramatic alterations of gene content or structure. These adaptations appear to include higher metabolic versatility and genomic plasticity to cope with the sparse and sporadic energy resources available, a preference for hydrophobic and smaller-volume amino acids in protein sequences, unique proteins not found in surface-dwelling species, and adaptations at the gene expression level. The deep-sea community is also characterized by a larger average genome size and a higher content of "selfish" genetic elements, such as transposases and prophages, whose propagation is apparently favored by more relaxed purifying (negative) selection in deeper waters.

NIH Grant Watch

BioTechniquesVol. 45, No. 3 NIH Grant WatchOpen AccessNIH Grant WatchK. Nybo† & B. Perry††K. Nybo††is assistant editor at BioTechniques.Search for more papers by this author & B. Perry††††is president of NIH Sales, Rockville, MD.Search for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000112941AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Self-assembling Peptides; Non-invasive Optical Brain ImagingBioTechniques and NIH Sales reviewed grants made by the National Institutes of Health during the last 30 days. Here are some highlights from among the largest 100 R01 and R03 grants awarded during July 2008.Self-assembling Peptides for Tissue Engineering $1,348,457(5R01EB003805-04, National Institute of Biomedical Imaging and Bioengineering) Alan Grodzinsky (Massachusetts Institute of Technology, Cambridge, MA)Goal: To design functional self-assembling peptide sequences for 3-D tissue engineering, measure and model the biophysics of the peptide self-assembly environment, and explore these environments in myocardium, cartilage, and liver. Experts in electrical, mechanical, and chemical engineering will collaborate with chemists, biologists, and clinical scientists to complete these aims.Non-invasive Optical Imaging of Neuropathology In Vivo $800,811(5R01AG026240-03, National Institute on Aging)Brian Bacskai (Massachusetts General Hospital, Boston, MA)Goal: To improve optical imaging techniques and generate novel contrast reagents for near-infrared molecular imaging. The multidisciplinary program includes advancement of the hardware and software of an existing optical imaging system and development of molecular imaging probes that target specific intracranial structures for in vivo imaging of neurological disease.Whole-genome Shotgun Sequencing Strategy and Assembly $759,485(5R01HG003474-04, National Human Genome Research Institute)David Jaffe (Massachusetts Institute of Technology, Cambridge, MA)Goal: To produce better quality genome sequences at lower cost by improving assembly of whole-genome shotgun sequencing data. This program will optimize strategy selection criteria for sequencing new organisms, develop algorithms for more accurate and complete genome assembly, and implement strict tests to maintain internal consistency.Molecular Analysis of Hotspots of Genetic Recombination $749,889(5R01GM031693-26A2, National Institute of General Medical Sciences)Gerald Smith (Fred Hutchinson Cancer Research Center, Seattle, WA)Goal: To understand the mechanism and regulation of recombination along chromosomes and across lifespan. This will be accomplished by studying hotspots of recombination in Escherichia coli and Schizosaccharomyces pombe to determine the proteins involved in recombinant structure formation and their interactions and to assess the roles of chromatin modifications in regulating recombination sites.The following six grants were awarded for the development of databases or software that may become valuable research tools for those working in the biosciences:Yeast Genetic Interaction Map $1,410,493(5R01HG002432-06, National Human Genome Research Institute)Jef Boeke (Johns Hopkins University, Baltimore, MD)Building a Framework for Exploration of Orthologs and Evolutionary Distances $84,500(5R03LM009261-02, National Library of Medicine)Dennis Wall (Harvard University Medical School, Boston, MA)Gene Expression Database for Mouse Development $1,017,606(5R01HD033745-11, National Institute of Child Health and Human Development)Martin Ringwald (Jackson Laboratory, Bar Harbor, ME)Comparative Toxicogenomics Database (CTD) $854,606(5R01ES014065-03, National Institute of Environmental Health Sciences)Carolyn Mattingly (Mount Desert Island Biological Lab, Salisbury Cove, ME)Dissemination of Cross-platform Software for Artifact Detection and Region of Interest $162,238(1R03EB008673-01, National Institute of Biomedical Imaging and Bioengineering)Satrajit Ghosh (Massachusetts Institute of Technology, Cambridge, MA)FiguresReferencesRelatedDetails Vol. 45, No. 3 Follow us on social media for the latest updates Metrics Downloaded 97 times History Published online 16 May 2018 Published in print September 2008 Information© 2008 Author(s)PDF download

An MCMC algorithm for haplotype assembly from whole-genome sequence data

In comparison to genotypes, knowledge about haplotypes (the combination of alleles present on a single chromosome) is much more useful for whole-genome association studies and for making inferences about human evolutionary history. Haplotypes are typically inferred from population genotype data using computational methods. Whole-genome sequence data represent a promising resource for constructing haplotypes spanning hundreds of kilobases for an individual. In this article, we propose a Markov chain Monte Carlo (MCMC) algorithm, HASH (haplotype assembly for single human), for assembling haplotypes from sequenced DNA fragments that have been mapped to a reference genome assembly. The transitions of the Markov chain are generated using min-cut computations on graphs derived from the sequenced fragments. We have applied our method to infer haplotypes using whole-genome shotgun sequence data from a recently sequenced human individual. The high sequence coverage and presence of mate pairs result in fairly long haplotypes (N50 length ~ 350 kb). Based on comparison of the sequenced fragments against the individual haplotypes, we demonstrate that the haplotypes for this individual inferred using HASH are significantly more accurate than the haplotypes estimated using a previously proposed greedy heuristic and a simple MCMC method. Using haplotypes from the HapMap project, we estimate the switch error rate of the haplotypes inferred using HASH to be quite low, ~1.1%. Our Markov chain Monte Carlo algorithm represents a general framework for haplotype assembly that can be applied to sequence data generated by other sequencing technologies. The code implementing the methods and the phased individual haplotypes can be downloaded from (http://www.cse.ucsd.edu/users/vibansal/HASH/).

Genome-wide analysis of microsatellite polymorphism in chicken circumventing the ascertainment bias

Studies of microsatellites evolution based on marker data almost inherently suffer from an ascertainment bias because there is selection for the most mutable and polymorphic loci during marker development. To circumvent this bias we took advantage of whole-genome shotgun sequence data from three unrelated chicken individuals that, when aligned to the genome reference sequence, give sequence information on two chromosomes from about one-fourth (375,000) of all microsatellite loci containing di- through pentanucleotide repeat motifs in the chicken genome. Polymorphism is seen at loci with as few as five repeat units, and the proportion of dimorphic loci then increases to 50% for sequences with approximately 10 repeat units, to reach a maximum of 75%-80% for sequences with 15 or more repeat units. For any given repeat length, polymorphism increases with decreasing GC content of repeat motifs for dinucleotides, nonhairpin-forming trinucleotides, and tetranucleotides. For trinucleotide repeats which are likely to form hairpin structures, polymorphism increases with increasing GC content, indicating that the relative stability of hairpins affects the rate of replication slippage. For any given repeat length, polymorphism is significantly lower for imperfect compared to perfect repeats and repeat interruptions occur in >15% of loci. However, interruptions are not randomly distributed within repeat arrays but are preferentially located toward the ends. There is negative correlation between microsatellite abundance and single nucleotide polymorphism (SNP) density, providing large-scale genomic support for the hypothesis that equilibrium microsatellite distributions are governed by a balance between rate of replication slippage and rate of point mutation.

End-sequencing and characterization of silkworm (Bombyx mori) bacterial artificial chromosome libraries

BackgroundWe performed large-scale bacterial artificial chromosome (BAC) end-sequencing of two BAC libraries (an EcoRI- and a BamHI-digested library) and conducted an in silico analysis to characterize the obtained sequence data, to make them a useful resource for genomic research on the silkworm (Bombyx mori).ResultsMore than 94000 BAC end sequences (BESs), comprising more than 55 Mbp and covering about 10.4% of the silkworm genome, were sequenced. Repeat-sequence analysis with known repeat sequences indicated that the long interspersed nuclear elements (LINEs) were abundant in BamHI BESs, whereas DNA-type elements were abundant in EcoRI BESs. Repeat-sequence analysis revealed that the abundance of LINEs might be due to a GC bias of the restriction sites and that the GC content of silkworm LINEs was higher than that of mammalian LINEs. In a BLAST-based sequence analysis of the BESs against two available whole-genome shotgun sequence data sets, more than 70% of the BESs had a BLAST hit with an identity of ≥ 99%. About 14% of EcoRI BESs and about 8% of BamHI BESs were paired-end clones with unique sequences at both ends. Cluster analysis of the BESs clarified the proportion of BESs containing protein-coding regions.ConclusionAs a result of this characterization, the identified BESs will be a valuable resource for genomic research on Bombyx mori, for example, as a base for construction of a BAC-based physical map. The use of multiple complementary BAC libraries constructed with different restriction enzymes also makes the BESs a more valuable genomic resource. The GenBank accession numbers of the obtained end sequences are DE283657–DE378560.

Organization and Evolution of Primate Centromeric DNA from Whole-Genome Shotgun Sequence Data

The major DNA constituent of primate centromeres is alpha satellite DNA. As much as 2%–5% of sequence generated as part of primate genome sequencing projects consists of this material, which is fragmented or not assembled as part of published genome sequences due to its highly repetitive nature. Here, we develop computational methods to rapidly recover and categorize alpha-satellite sequences from previously uncharacterized whole-genome shotgun sequence data. We present an algorithm to computationally predict potential higher-order array structure based on paired-end sequence data and then experimentally validate its organization and distribution by experimental analyses. Using whole-genome shotgun data from the human, chimpanzee, and macaque genomes, we examine the phylogenetic relationship of these sequences and provide further support for a model for their evolution and mutation over the last 25 million years. Our results confirm fundamental differences in the dispersal and evolution of centromeric satellites in the Old World monkey and ape lineages of evolution.

Highly efficient concerted evolution in the ribosomal DNA repeats: Total rDNA repeat variation revealed by whole-genome shotgun sequence data

Repeat families within genomes are often maintained with similar sequences. Traditionally, this has been explained by concerted evolution, where repeats in an array evolve "in concert" with the same sequence via continual turnover of repeats by recombination. Another form of evolution, birth-and-death evolution, can also explain this pattern, although in this case selection is the critical force maintaining the repeats. The level of intragenomic variation is the key difference between these two forms of evolution. The prohibitive size and repetitive nature of large repeat arrays have made determination of the absolute level of intragenomic repeat variability difficult, thus there is little evidence to support concerted evolution over birth-and-death evolution for many large repeat arrays. Here we use whole-genome shotgun sequence data from the genome projects of five fungal species to reveal absolute levels of sequence variation within the ribosomal RNA gene repeats (rDNA). The level of sequence variation is remarkably low. Furthermore, the polymorphisms that are detected are not functionally constrained and seem to exist beneath the level of selection. These results suggest the rDNA is evolving via concerted evolution. Comparisons with a repeat array undergoing birth-and-death evolution provide a clear contrast in the level of repeat array variation between these two forms of evolution, confirming that the rDNA indeed does evolve via concerted evolution. These low levels of intra-genomic variation are consistent with a model of concerted evolution in which homogenization is very rapid and efficiently maintains highly similar repeat arrays.

Molecular characterisation of SALMFamide neuropeptides in sea urchins

The SALMFamides are a family of neuropeptides found in species belonging to the phylum Echinodermata. Members of this family have been identified in starfish (class Asteroidea) and in sea cucumbers (class Holothuroidea) but not in other echinoderms. Our aim here was to characterise SALMFamide neuropeptides in sea urchins (class Echinoidea). Radioimmunoassays for the starfish SALMFamides S1 and S2 were used to test for related peptides in whole-body acetone extracts of the sea urchin Echinus esculentus. Fractionation of extracts using high performance liquid chromatography (HPLC) revealed several peaks of SALMFamide-like immunoreactivity, with two S2-like immunoreactive peaks (3 and 4) being the most prominent. However, peak 4 could not be purified to homogeneity and although peak 3 was purified, only a partial sequence (MRYH) could be obtained. An alternative strategy for identification of echinoid SALMFamides was provided by sequencing the genome of the sea urchin Strongylocentrotus purpuratus. Analysis of whole-genome shotgun sequence data using the Basic Local Alignment Search Tool (BLAST) identified a contig (347664) that contains a coding region for seven putative SALMFamide neuropeptides (PPVTTRSKFTFamide, DAYSAFSFamide, GMSAFSFamide, AQPSFAFamide, GLMPSFAFamide, PHGGSAFVFamide and GDLAFAFamide), which we have named SpurS1-SpurS7, respectively. Three of these peptides (SpurS1-3) have the C-terminal sequences TFamide or SFamide, which are identical or similar to the C-terminal region of the starfish SALMFamide S2. This may explain the occurrence of several S2-like immunoreactive peptides in extracts of Echinus esculentus. Detailed analysis of the sequence of contig 347664 indicated that the SALMFamide gene in Strongylocentrotus purpuratus comprises two exons, with the first exon encoding a signal peptide sequence and the second exon encoding SpurS1-SpurS7. Characterisation of this gene is important because it is the first echinoderm neuropeptide precursor sequence to be identified and, more specifically, it provides our first insight into the structure and organisation of a SALMFamide gene in an echinoderm. In particular, it has revealed a hitherto unknown complexity in the diversity of SALMFamide neuropeptides that may occur in an echinoderm species because all previous studies, which relied on peptide purification and sequencing, revealed only two SALMFamide neuropeptides in each species examined. It now remains to be established whether or not the occurrence of more than two SALMFamides in Strongylocentrotus purpuratus is a feature that is peculiar to this species and to echinoids in general or is more widespread across the phylum Echinodermata. Identification of SpurS1-SpurS7 provides the basis for comparative analysis of the physiological actions of these peptides in sea urchins and for exploitation of the sea urchin genome sequence to identify the receptor(s) that mediate effects of SALMFamides in echinoderms.