Assessment of the relationship between signal intensities and transcript concentration for Affymetrix GeneChip®arrays

Background

The availability of both mouse and human draft genomes has marked the beginning of a new era of comparative mammalian genomics. The two available mouse genome assemblies, from the public mouse genome sequencing consortium and Celera Genomics, were obtained using different clone libraries and different assembly methods.

Results

We present here a critical comparison of the two latest mouse genome assemblies. The utility of the combined genomes is further demonstrated by comparing them with the human 'golden path' and through a subsequent analysis of a resulting conserved sequence element (CSE) database, which allows us to identify over 6,000 potential novel genes and to derive independent estimates of the number of human protein-coding genes.

Conclusion

The Celera and public mouse assemblies differ in about 10% of the mouse genome. Each assembly has advantages over the other: Celera has higher accuracy in base-pairs and overall higher coverage of the genome; the public assembly, however, has higher sequence quality in some newly finished bacterial artifical chromosome clone (BAC) regions and the data are freely accessible. Perhaps most important, by combining both assemblies, we can get a better annotation of the human genome; in particular, we can obtain the most complete set of CSEs, one third of which are related to known genes and some others are related to other functional genomic regions. More than half the CSEs are of unknown function. From the CSEs, we estimate the total number of human protein-coding genes to be about 40,000. This searchable publicly available online CSEdb will expedite new discoveries through comparative genomics.     

Y chromosome and other heterochromatic sequences of the Drosophila melanogaster genome: how far can we go?

Whole genome shotgun assemblies have proven remarkably successful in reconstructing the bulk of euchromatic genes, with the only limit appearing to be determined by the sequencing depth. For genes imbedded in heterochromatin, however, the low cloning efficiency of repetitive sequences, combined with the computational challenges, demand that additional clues be used to annotate the sequences. One approach that has proven very successful in identifying protein coding genes in Y-linked heterochromatin of Drosophila melanogaster has been to make a BLASTable database of the small, unmapped contigs and fragments leftover at the end of a shotgun assembly, and to attempt to capture these by blasting with an appropriate query sequence. This approach often yields a staggered alignment of contigs from the unmapped set to the query sequence, as though the disjoint contigs represent small portions of the gene. Further inspection frequently shows that the contigs are broken by very large, heterochromatic introns. Methods of this sort are being expanded to make best use of all available clues to determine which unmapped contigs are associated with genes. These include use of EST libraries, and, in the case of the Y chromosome, testing of male specific genes and reduced shotgun depth of relevant contigs. It appears much more hopeful than anyone would have imagined that whole genome shotgun assemblies can recover the great bulk of even heterochromatic genes.     

Design of a compartmentalized shotgun assembler for the human genome

Two different strategies for determining the human genome are currently being pursued: one is the "clone-by-clone" approach, employed by the publicly funded project, and the other is the "whole genome shotgun assembler" approach, favored by researchers at Celera Genomics. An interim strategy employed at Celera, called compartmentalized shotgun assembly, makes use of preliminary data produced by both approaches. In this paper we describe the design, implementation and operation of the "compartmentalized shotgun assembler".     

A preprocessor for shotgun assembly of large genomes.

The whole-genome shotgun (WGS) assembly technique has been remarkably successful in efforts to determine the sequence of bases that make up a genome. WGS assembly begins with a large collection of short fragments that have been selected at random from a genome. The sequence of bases at each end of the fragment is determined, albeit imprecisely, resulting in a sequence of letters called a "read." Each letter in a read is assigned a quality value, which estimates the probability that a sequencing error occurred in determining that letter. Reads are typically cut off after about 500 letters, where sequencing errors become endemic. We report on a set of procedures that (1) corrects most of the sequencing errors, (2) changes quality values accordingly, and (3) produces a list of "overlaps," i.e., pairs of reads that plausibly come from overlapping parts of the genome. Our procedures, which we call collectively the "UMD Overlapper," can be run iteratively and as a preprocessor for other assemblers. We tested the UMD Overlapper on Celera's Drosophila reads. When we replaced Celera's overlap procedures in the front end of their assembler, it was able to produce a significantly improved genome.     

Genome assembly has a major impact on gene content: a comparison of annotation in two Bos taurus assemblies

Gene and SNP annotation are among the first and most important steps in analyzing a genome. As the number of sequenced genomes continues to grow, a key question is: how does the quality of the assembled sequence affect the annotations? We compared the gene and SNP annotations for two different Bos taurus genome assemblies built from the same data but with significant improvements in the later assembly. The same annotation software was used for annotating both sequences. While some annotation differences are expected even between high-quality assemblies such as these, we found that a staggering 40% of the genes (>9,500) varied significantly between assemblies, due in part to the availability of new gene evidence but primarily to genome mis-assembly events and local sequence variations. For instance, although the later assembly is generally superior, 660 protein coding genes in the earlier assembly are entirely missing from the later genome''s annotation, and approximately 3,600 (15%) of the genes have complex structural differences between the two assemblies. In addition, 12–20% of the predicted proteins in both assemblies have relatively large sequence differences when compared to their RefSeq models, and 6–15% of bovine dbSNP records are unrecoverable in the two assemblies. Our findings highlight the consequences of genome assembly quality on gene and SNP annotation and argue for continued improvements in any draft genome sequence. We also found that tracking a gene between different assemblies of the same genome is surprisingly difficult, due to the numerous changes, both small and large, that occur in some genes. As a side benefit, our analyses helped us identify many specific loci for improvement in the Bos taurus genome assembly.     

MGView: an alignment and visualization tool to enhance gap closure of microbial genomes

Gap closure is a challenging phase in microbial random shotgun genome sequencing projects, particularly since genome assemblies are often complicated by the presence of repeat elements, insertion sequences and other similar factors that contribute to sequence misassemblies. While it is well recognized that the conservation of genetic information between microbial genomes, combined with the exponential increase in available microbial sequences, can be exploited to increase the efficiency of gap closure, we lack the computational tools to aid in this process. We describe here a new tool, MGView, which was developed to create a graphical depiction of the alignment of a set of microbial contigs against a completed microbial genome. The results of our assembly of the Staphylococcus aureus RF122 genome show that MGView enables a considerable reduction in time and economic cost associated with closure. Together, the results also show that the application of MGView not only enables a reduction in fold-coverage requirements of the random shotgun sequence phase, but also provides interesting insights into differences in gene content and organization between finished and unfinished microbial genomes.     

Management and visualization of whole genome shotgun assemblies using SAM

We have designed and implemented a system to manage whole genome shotgun sequences and whole genome sequence assembly data flow. The Sequence Assembly Manager (SAM) consists primarily of a MySQL relational database and Perl applications designed to easily manipulate and coordinate the analysis of sequence information and to view and report genome assembly progress through its Common Gateway Interface (CGI) web interface. The application includes a tool to compare sequence assemblies to fingerprint maps that has been used successfully to improve and validate both maps and sequence assemblies of the Rhodococcus sp.RHAI and Cryptococcus neoformans WM276 genomes.     

Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Hierarchical shotgun sequencing remains the method of choice for assembling high‐quality reference sequences of complex plant genomes. The efficient exploitation of current high‐throughput technologies and powerful computational facilities for large‐insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole‐genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high‐quality assemblies of a large number of clones to assemble map‐based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.     

An integrated physical, genetic and cytogenetic map of Brachypodium distachyon, a model system for grass research

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent validation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation.     

Whole-genome sequencing and assembly with high-throughput, short-read technologies

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology.     

Efficiently detecting polymorphisms during the fragment assembly process

MOTIVATION: Current genomic sequence assemblers assume that the input data is derived from a single, homogeneous source. However, recent whole-genome shotgun sequencing projects have violated this assumption, resulting in input fragments covering the same region of the genome whose sequences differ due to polymorphic variation in the population. While single-nucleotide polymorphisms (SNPs) do not pose a significant problem to state-of-the-art assembly methods, these methods do not handle insertion/deletion (indel) polymorphisms of more than a few bases. RESULTS: This paper describes an efficient method for detecting sequence discrepencies due to polymorphism that avoids resorting to global use of more costly, less stringent affine sequence alignments. Instead, the algorithm uses graph-based methods to determine the small set of fragments involved in each polymorphism and performs more sophisticated alignments only among fragments in that set. Results from the incorporation of this method into the Celera Assembler are reported for the D. melanogaster, H. sapiens, and M. musculus genomes.     

Computational comparison of two mouse draft genomes and the human golden path

Background  

The availability of both mouse and human draft genomes has marked the beginning of a new era of comparative mammalian genomics. The two available mouse genome assemblies, from the public mouse genome sequencing consortium and Celera Genomics, were obtained using different clone libraries and different assembly methods.     

The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole‐genome shotgun sequencing of the nuclear genome of flax. Seven paired‐end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep‐coverage (approximately 94× raw, approximately 69× filtered) short‐sequence reads (44–100 bp), produced a set of scaffolds with N₅₀ = 694 kb, including contigs with N₅₀ = 20.1 kb. The contig assembly contained 302 Mb of non‐redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole‐genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis‐assembly of regions at the genome scale. A total of 43 384 protein‐coding genes were predicted in the whole‐genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K_s) observed within duplicate gene pairs was consistent with a recent (5–9 MYA) whole‐genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam‐A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole‐genome shotgun short‐sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.     

Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ)

Next‐generation whole‐genome shotgun assemblies of complex genomes are highly useful, but fail to link nearby sequence contigs with each other or provide a linear order of contigs along individual chromosomes. Here, we introduce a strategy based on sequencing progeny of a segregating population that allows de novo production of a genetically anchored linear assembly of the gene space of an organism. We demonstrate the power of the approach by reconstructing the chromosomal organization of the gene space of barley, a large, complex and highly repetitive 5.1 Gb genome. We evaluate the robustness of the new assembly by comparison to a recently released physical and genetic framework of the barley genome, and to various genetically ordered sequence‐based genotypic datasets. The method is independent of the need for any prior sequence resources, and will enable rapid and cost‐efficient establishment of powerful genomic information for many species.     

Using shotgun sequence data to find active restriction enzyme genes

Whole genome shotgun sequence analysis has become the standard method for beginning to determine a genome sequence. The preparation of the shotgun sequence clones is, in fact, a biological experiment. It determines which segments of the genome can be cloned into Escherichia coli and which cannot. By analyzing the complete set of sequences from such an experiment, it is possible to identify genes lethal to E. coli. Among this set are genes encoding restriction enzymes which, when active in E. coli, lead to cell death by cleaving the E. coli genome at the restriction enzyme recognition sites. By analyzing shotgun sequence data sets we show that this is a reliable method to detect active restriction enzyme genes in newly sequenced genomes, thereby facilitating functional annotation. Active restriction enzyme genes have been identified, and their activity demonstrated biochemically, in the sequenced genomes of Methanocaldococcus jannaschii, Bacillus cereus ATCC 10987 and Methylococcus capsulatus.     

Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies

Background

The size and complexity of conifer genomes has, until now, prevented full genome sequencing and assembly. The large research community and economic importance of loblolly pine, Pinus taeda L., made it an early candidate for reference sequence determination.

Results

We develop a novel strategy to sequence the genome of loblolly pine that combines unique aspects of pine reproductive biology and genome assembly methodology. We use a whole genome shotgun approach relying primarily on next generation sequence generated from a single haploid seed megagametophyte from a loblolly pine tree, 20-1010, that has been used in industrial forest tree breeding. The resulting sequence and assembly was used to generate a draft genome spanning 23.2 Gbp and containing 20.1 Gbp with an N50 scaffold size of 66.9 kbp, making it a significant improvement over available conifer genomes. The long scaffold lengths allow the annotation of 50,172 gene models with intron lengths averaging over 2.7 kbp and sometimes exceeding 100 kbp in length. Analysis of orthologous gene sets identifies gene families that may be unique to conifers. We further characterize and expand the existing repeat library based on the de novo analysis of the repetitive content, estimated to encompass 82% of the genome.

Conclusions

In addition to its value as a resource for researchers and breeders, the loblolly pine genome sequence and assembly reported here demonstrates a novel approach to sequencing the large and complex genomes of this important group of plants that can now be widely applied.