Paul Predki

An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium.

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple developmental alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9 Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.

The DNA sequence and biology of human chromosome 19

Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 is an α-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Photosynthesis may be photoheterotrophic using organic compounds as both a carbon and a reducing source, or photoautotrophic using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. The structural components of this metabolically diverse organism and their modes of integrated regulation are encoded by a genome of ∼4.5 Mb in size. The genome comprises two chromosomes CI and CII (2.9 and 0.9 Mb, respectively) and five other replicons. Sequencing of the genome has been carried out by two groups, the Joint Genome Institute, which carried out shotgun-sequencing of the entire genome and The University of Texas-Houston Medical School, which carried out a targeted sequencing strategy of CII. Here we describe our current understanding of the genome when data from both of these groups are combined. Previous work had suggested that the two chromosomes are equal partners sharing responsibilities for fundamental cellular processes. This view has been reinforced by our preliminary analysis of the virtually completed genome sequence. We also have some evidence to suggest that two of the plasmids, pRS241a and pRS241b encode chromosomal type functions and their role may be more than that of accessory elements, perhaps representing replicons in a transition state.

Whole-genome comparative analysis of three phytopathogenic Xylella fastidiosa strains

Xylella fastidiosa (Xf) causes wilt disease in plants and is responsible for major economic and crop losses globally. Owing to the public importance of this phytopathogen we embarked on a comparative analysis of the complete genome of Xf pv citrus and the partial genomes of two recently sequenced strains of this species: Xf pv almond and Xf pv oleander, which cause leaf scorch in almond and oleander plants, respectively. We report a reanalysis of the previously sequenced Xf 9a5c (CVC, citrus) strain and the two “gapped” Xf genomes revealing ORFs encoding critical functions in pathogenicity and conjugative transfer. Second, a detailed whole-genome functional comparison was based on the three sequenced Xf strains, identifying the unique genes present in each strain, in addition to those shared between strains. Third, an “in silico” cellular reconstruction of these organisms was made, based on a comparison of their core functional subsystems that led to a characterization of their conjugative transfer machinery, identification of potential differences in their adhesion mechanisms, and highlighting of the absence of a classical quorum-sensing mechanism. This study demonstrates the effectiveness of comparative analysis strategies in the interpretation of genomes that are closely related.

The complete sequence of human chromosome 5

Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

On the High Value of Low Standards

Is there a case to be made for draft sequencing? First, we need to get a fix on how much less it costs than complete genome sequencing, how much faster and/or easier it is to do, and how much and what types of scientific utility are sacrificed. But this is not a straightforward issue. No accepted standard for draft sequence data exists; in current practice it ranges from ∼3-fold coverage in short ( 600-bp), “paired-end” (PE) reads (sequencing reads are taken from both ends of the insert in a double-stranded vector and therefore come in oppositely directed pairs separated by an approximately known distance) of mixed separation lengths. Quality differences over that spectrum are relatively great, as are, though to a much smaller extent, cost differences. The “draft-or-finish” alternatives are hardly exclusive; mixed, staged, or context-dependent strategies may also make sense. All the parameters are evolving rapidly. And finally, there is as yet too little experience to support definitive answers, although clearly enough to get an argument going in the better genome bars. First, we address the production side of the question; consider the hypothetical case of sequencing factory X. This exemplary facility can produce over 30 Mb of high-quality (PE) bases per day at a fully loaded marginal cost of 0.3¢ base. Factory X has concluded that for most DNA, 8× PE coverage is usually optimal, both for producing draft data that are not intended for subsequent finishing and as a substrate for finishing. With this choice, finish-ready draft data have, at factory X, a current marginal cost of ∼2.5¢ base and can be produced at a rate of 3.6 Mb/day with a delay from time of DNA receipt to draft product on the order of 2 weeks. The quality of this sequence is discussed below, but the general nature of its coverage integrity should be noted here. In ∼8-fold PE draft data, the overall coverage is typically high (>95% of the sequence represented). Most importantly, and especially so if a judicious mix of large and small inserts is used in the sequencing, “almost all” points in the sequence—including gaps between the contigs (contigs are contiguous stretches of sequence produced by assembling overlapping individual reads)—are bridged, or spanned, by multiple plasmid clones. This permits the automatic production of relatively high-quality, internally verified assembly and makes it possible to order and orient most of the contigs relative to each other to form large “scaffolds,” or sequence islands of valid order and high coverage. In such data, the expected error rate across genes is often better than 1/104, and a good estimate of the accuracy of each base can be made available. Factory X can also finish such data to full “Bermuda” standards, i.e., an expected base-calling error rate of <1/104 and no gaps or other errors that mortal efforts could remove (these standards were established at meetings of the international Human Genome Project community), for an average additional cost of 7¢ base (and thus for a total cost of ∼10¢ base). Somewhat typically, however, factory Xs finishing capacity is manyfold below its drafting capacity. Furthermore, the time needed to finish a segment of draft sequence can average several months and is highly variable. In this landscape, “full Bermuda” data are about four times as expensive, and very much slower to produce, than “high-quality” draft data. For the extra cost of finishing a bacterial genome, three additional ones could be drafted. While factory X is finishing a bacterial genome, it could draft, in the sense described, upwards of a hundred more. To our necessarily imperfect knowledge, no sequencing facility is currently producing either PE raw data or “fully finished” sequence data for true costs significantly below those quoted. But the relative advantage in cost and project completion time of draft versus finished sequence data at factory X might well not be the same in other facilities. And of course, the differences in steady-state production capacity for draft versus finished sequence used in the example are in large measure merely an arbitrary matter of resource commitment. Also, there are some, at least potential, hidden costs in producing draft data that should be considered. (i) Draft sequence errors and imperfections may mislead users and thereby entail costs in wasted effort and delay. (ii) It may be substantially more expensive on average to finish draft sequence data later, should it prove desirable, than to do so at the start and in the same laboratory. (iii) Many have seen a risk that the will (at either the funding or bench level) to ever fully finish sequence data will be lost should we permit ourselves the cheap and easy pleasures of draft sequencing. We comment a little on these questions at the end. The next issue is the quality and utility of draft sequence data, focusing in particular on what we know about (i) sequence coverage, (ii) gene recovery and quality, and (iii) chromosome integrity and long-range order.

Protein Microarray-Based Screening of Antibody Specificity

The increased use of antibodies as therapeutics, as well as the growing demand for large numbers of antibodies for high-throughput protein analyses, has been accompanied by a need for more specific antibodies. An array containing every protein for the relevant organism represents the ideal format for an assay to test antibody specificity since it allows the simultaneous screening of thousands of proteins in relatively normalized quantities. Indeed, the use of a yeast proleome array to profile the specificity of several antibodies directed against yeast proteins has recently been described. In this chapter, we present a detailed description of the methods used to probe protein arrays with antibodies as well as the technical issues to consider when carrying out such experiments.

Practical applications of rolling circle amplification of DNA templates.

Since its recent implementation at one of the worlds largest high-throughput sequencing centers, the utility of MP-RCA for DNA sequencing has been thoroughly validated. However, applications of this technology extend far beyond DNA sequencing. While many of these applications have been explored in this chapter, the future will undoubtedly add to this growing list.