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".

Comparison of papillomavirus and immunodeficiency virus evolutionary patterns in the context of a papillomavirus vaccine.

In contemplating a vaccine for human papillomaviruses (HPVs), it is important to consider the evolutionary context in which such a vaccine would be deployed. The human immunodeficiency virus, having been the subject of even more extensive study than HPV, shares certain salient features with regards to phylogenetic structure, and may serve as a model for contemplation of possible difficulties with HPV vaccination. However, there are also striking differences in the evolutionary potentials and histories of the viruses that permit an optimistic outlook for HPV. These similarities and differences, as well as their implications for vaccination studies, are reviewed.

Multiple-changepoint testing for an alternating segments model of a binary sequence.

A binary sequence may give the appearance of being composed of alternating segments with relatively high and relatively low probability of success. Determining whether such an alternating pattern is significant is a multiple-changepoint problem where the number of segments and their success probabilities are unknown, with the added constraint of segment alternation. A dynamic programming method for determining the optimal segmentation into a given number of segments is provided. Given this, a variation on the simulation method of Venter and Steel (1996, Computational Statistics and Data Analysis 22, 481-504) may be employed to test the null hypothesis of a homogeneous sequence as well as to estimate the number and location of changepoints. A sample application, the assessment of the possibility of genetic recombination in HIV sequences, is presented.

Large-scale comparison of fungal sequence information: mechanisms of innovation in Neurospora crassa and gene loss in Saccharomyces cerevisiae.

We report a large-scale comparison of sequence data from the filamentous fungus Neurospora crassa with the complete genome sequence of Saccharomyces cerevisiae. N. crassa is considerably more morphologically and developmentally complex than S. cerevisiae. We found that N. crassa has a much higher proportion of "orphan" genes than S. cerevisiae, suggesting that its morphological complexity reflects the acquisition or maintenance of novel genes, consistent with its larger genome. Our results also indicate the loss of specific genes from S. cerevisiae. Surprisingly, some of the genes lost from S. cerevisiae are involved in basic cellular processes, including translation and ion (especially calcium) homeostasis. Horizontal gene transfer from prokaryotes appears to have played a relatively modest role in the evolution of the N. crassa genome. Differences in the overall rate of molecular evolution between N. crassa and S. cerevisiae were not detected. Our results indicate that the current public sequence databases have fairly complete samples of gene families with ancient conserved regions, suggesting that further sequencing will not substantially change the proportion of genes with homologs among distantly related groups. Models of the evolution of fungal genomes compatible with these results, and their functional implications, are discussed.

A whole-genome assembly of Drosophila.

We report on the quality of a whole-genome assembly of Drosophila melanogaster and the nature of the computer algorithms that accomplished it. Three independent external data sources essentially agree with and support the assembly's sequence and ordering of contigs across the euchromatic portion of the genome. In addition, there are isolated contigs that we believe represent nonrepetitive pockets within the heterochromatin of the centromeres. Comparison with a previously sequenced 2.9- megabase region indicates that sequencing accuracy within nonrepetitive segments is greater than 99. 99% without manual curation. As such, this initial reconstruction of the Drosophila sequence should be of substantial value to the scientific community.

Weighted neighbor joining: a likelihood-based approach to distance-based phylogeny reconstruction.

We introduce a distance-based phylogeny reconstruction method called "weighted neighbor joining," or "Weighbor" for short. As in neighbor joining, two taxa are joined in each iteration; however, the Weighbor criterion for choosing a pair of taxa to join takes into account that errors in distance estimates are exponentially larger for longer distances. The criterion embodies a likelihood function on the distances, which are modeled as correlated Gaussian random variables with different means and variances, computed under a probabilistic model for sequence evolution. The Weighbor criterion consists of two terms, an additivity term and a positivity term, that quantify the implications of joining the pair. The first term evaluates deviations from additivity of the implied external branches, while the second term evaluates confidence that the implied internal branch has a positive branch length. Compared with maximum-likelihood phylogeny reconstruction, Weighbor is much faster, while building trees that are qualitatively and quantitatively similar. Weighbor appears to be relatively immune to the "long branches attract" and "long branch distracts" drawbacks observed with neighbor joining, BIONJ, and parsimony.

The psychiatrist's dilemma: a conflict of roles in legal executions.

In the United States, a critical controversy is taking place in regard to psychiatrists' and other physicians' participation in legal executions. Under pressure from the criminal justice system and legislatures to expedite executions, some forensic psychiatrists have succeeded in loosening traditional prohibitions against such participation. Further, there has been a weakening of the prohibition against treatment designed to facilitate immediate execution of those condemned to death. The rationale offered for these departures from current psychiatric ethical codes is the novel notion that when a psychiatrist acts in the court or criminal justice situation, that individual is no longer a psychiatrist and is not bound by psychiatric ethics. Rather, the forensic psychiatrist, termed a 'forensicist', serves as an assistant in the 'administration of justice' or 'an agent of the State' and thus works in a different ethical framework from the ordinary psychiatrist. This justification has similarities to the rationale offered by physicians involved in human experiments and other criminal acts in Nazi Germany, as well as psychiatrists in the former Soviet Union who explained their involvement in psychiatric abuse as a result of being agents of the State and thus not responsible for carrying out orders. Clearly, this controversy could be eliminated by a campaign for the abolition of capital punishment, characterised by the American Psychiatric Association as 'anachronistic, brutalizing [and] ineffective'. Such a campaign should serve as a call for psychiatrists and other physicians to join in the struggle to uphold ethical and moral principles.

Minimally selected p and other tests for a single abrupt changepoint in a binary sequence.

A novel changepoint statistic based on the minimum value, over possible changepoint locations, of Fisher's Exact Test, is introduced. Specific points in the exact distribution of the minimally selected Fisher's value may be rapidly calculated as a lattice-path counting problem via known recurrence methods. The test is compared to the Kolmogorov-Smirnov two-sample test, the maximally selected chi-square, and a likelihood ratio test. The tests are applied to assessing recombination in genetic sequences of HIV.

Evolutionary distances for protein-coding sequences: modeling site-specific residue frequencies.

Estimation of evolutionary distances from coding sequences must take into account protein-level selection to avoid relative underestimation of longer evolutionary distances. Current modeling of selection via site-to-site rate heterogeneity generally neglects another aspect of selection, namely position-specific amino acid frequencies. These frequencies determine the maximum dissimilarity expected for highly diverged but functionally and structurally conserved sequences, and hence are crucial for estimating long distances. We introduce a codon-level model of coding sequence evolution in which position-specific amino acid frequencies are free parameters. In our implementation, these are estimated from an alignment using methods described previously. We use simulations to demonstrate the importance and feasibility of modeling such behavior; our model produces linear distance estimates over a wide range of distances, while several alternative models underestimate long distances relative to short distances. Site-to-site differences in rates, as well as synonymous/nonsynonymous and first/second/third-codon-position differences, arise as a natural consequence of the site-to-site differences in amino acid frequencies.

Human papillomavirus type 16 variant lineages in United States populations characterized by nucleotide sequence analysis of the E6, L2, and L1 coding segments.

Human papillomavirus type 16 (HPV16) nucleotide sequence variations in the E6 (nucleotide positions [nt] 104 to 559), L2 (nt 4272 to 5657), and L1 (nt 5665 to 7148) open reading frames (ORFs), and the long control region (nt 7479 to 7842), were examined in 29 selected United States isolates. Of 3,690 nucleotide positions, 129 (3.5%) varied. The maximum pairwise distance was 66 nucleotide differences, or 1.8%. Nucleotide variations within different genome segments were phylogenetically compatible, and nucleotide changes within E6, L2, and L1 contained phylogenetic information beyond that provided in the long control region. Most isolates were classified as members of HPV16 lineages that have been described previously. However, two novel phylogenetic branches were identified. The L2 ORF was the most variable coding segment. L2 synonymous and nonsynonymous nucleotide changes were distributed asymmetrically. The amino-terminal half of the L2 protein was remarkably conserved among all isolates, suggesting that the region is under evolutionary constraint. The amino-terminal region of the E6 ORF was relatively varied, especially at E6 amino acid positions 10 and 14. Several amino acid difference in the L1 ORF were observed between lineages. Forty-nine amino acid variations across all sequenced coding regions were observed. These amino acid differences may be relevant to differences in the generation of humoral or cell-mediated immune responses to HPV16 variants. Our data form a basis for considering HPV16 sequence variation in the rational design of vaccine strategies and as an epidemiologic correlate of cervical cancer risk.

Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy.

Our aim was to study the phylogenetic relationships of all known papillomaviruses (PVs) and the possibility of establishing a supratype taxonomic classification based on this information. Of the many detectably homologous segments present in PV genomes, a 291-bp segment of the L1 gene is notable because it is flanked by the MY09 and MY11 consensus primers and contains highly conserved amino acid residues which simplify sequence alignment. We determined the MY09-MY11 sequences of human PV type 20 (HPV-20), HPV-21, HPV-22, HPV-23, HPV-24, HPV-36, HPV-37, HPV-38, HPV-48, HPV-50, HPV-60, HPV-70, HPV-72, HPV-73, ovine (sheep) PV, bovine PV type 3 (BPV-3), BPV-5, and BPV-6 and created a database which now encompasses HPV-1 to HPV-70, HPV-72, HPV-73, seven yet untyped HPV genomes, and 15 animal PV types. Three additional animal PVs were analyzed on the basis of other sequence data. We constructed phylogenies based on partial L1 and E6 gene sequences and distinguished five major clades that we call supergroups. One of them unites 54 genital PV types, which can be further divided into eleven groups. The second supergroup has 24 types and unites most PVs that are typically found in epidermodysplasia verruciformis patients but also includes several types typical of other cutaneous lesions, like HPV-4. The third supergroup unites the six known ungulate fibropapillomaviruses, the fourth includes the cutaneous ungulate PVs BPV-3, BPV-4, and BPV-6, and the fifth includes HPV-1, HPV-41, HPV-63, the canine oral PV, and the cottontail rabbit PV. The chaffinch PV and two rodent PVs, Micromys minutus PV and Mastomys natalensis PV, are left ungrouped because of the relative isolation of each of their lineages. Within most supergroups, groups formed on the basis of cladistic principles unite phenotypically similar PV types. We discuss the basis of our classification, the concept of the PV type, speciation, PV-host evolution, and estimates of their rates of evolution.