Demography-adjusted tests of neutrality based on genome-wide SNP data.

Tests of the neutral evolution hypothesis are usually built on the standard null model which assumes that mutations are neutral and the population size remains constant over time. However, it is unclear how such tests are affected if the last assumption is dropped. Here, we extend the unifying framework for tests based on the site frequency spectrum, introduced by Achaz and Ferretti, to populations of varying size. Key ingredients are the first two moments of the site frequency spectrum. We show how these moments can be computed analytically if a population has experienced two instantaneous size changes in the past. We apply our method to data from ten human populations gathered in the 1000 genomes project, estimate their demographies and define demography-adjusted versions of Tajima's D, Fay & Wu's H, and Zeng's E. Our results show that demography-adjusted test statistics facilitate the direct comparison between populations and that most of the differences among populations seen in the original unadjusted tests can be explained by their underlying demographies. Upon carrying out whole-genome screens for deviations from neutrality, we identify candidate regions of recent positive selection. We provide track files with values of the adjusted and unadjusted tests for upload to the UCSC genome browser.

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis.

Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.

SGP-1: prediction and validation of homologous genes based on sequence alignments.

Conventional methods of gene prediction rely on the recognition of DNA-sequence signals, the coding potential or the comparison of a genomic sequence with a cDNA, EST, or protein database. Reasons for limited accuracy in many circumstances are species-specific training and the incompleteness of reference databases. Lately, comparative genome analysis has attracted increasing attention. Several analysis tools that are based on human/mouse comparisons are already available. Here, we present a program for the prediction of protein-coding genes, termed SGP-1 (Syntenic Gene Prediction), which is based on the similarity of homologous genomic sequences. In contrast to most existing tools, the accuracy of depends little on species-specific properties such as codon usage or the nucleotide distribution. may therefore be applied to nonstandard model organisms in vertebrates as well as in plants, without the need for extensive parameter training. In addition to predicting genes in large-scale genomic sequences, the program may be useful to validate gene structure annotations from databases. To this end, SGP-1 output also contains comparisons between predicted and annotated gene structures in HTML format. The program can be accessed via a Web server at http://soft.ice.mpg.de/sgp-1. The source code, written in ANSI C, is available on request from the authors.

Statistics of divergence times.

Given the number of nucleotide substitutions between two species (K) and the substitution rate nu, the expectation of the corresponding divergence time is usually calculated as K/(2 nu). This is strictly true only if nu is regarded as a constant because the ratio of two random variables, such as K/(2 nu), has distributional properties different from those of the distribution of K. Therefore, both the mean and any confidence interval for divergence times are unknown in this situation. We model the distribution of K and nu using the Gamma distribution and calculate the mean and 95% confidence interval for the corresponding divergence time. These calculations are compared with results obtained by bootstrapping sequence data from the model plant Arabidopsis thaliana and its relatives. We show that for nonoverlapping pairs of phylogenetic distances, our method approaches the bootstrap results very closely. In contrast, regarding the mutation rate as a constant leads to strong underestimation of the confidence interval. An implementation of our method of computing divergence times is accessible through a web interface at http://www.soft.ice.mpg.de/cite.

Distinguishing recombination and intragenic gene conversion by linkage disequilibrium patterns.

Deterministic theory suggests that reciprocal recombination and intragenic, interallelic conversion have different effects on the linkage disequilibrium between a pair of genetic markers. Under a model of reciprocal recombination, the decay rate of linkage disequilibrium depends on the distance between the two markers, while under conversion the decay rate is independent of this distance, provided that conversion tracts are short. A population genetic three-locus model provides a function Q of two-locus linkage disequilibria. Viewed as a random variable, Q is the basis for a test of the relative impact of conversion and recombination. This test requires haplotype frequency data of a sufficiently variable three-locus system. One of the few examples currently available is data from the Human Leukocyte Antigen (HLA) class I genes of three Amerindian populations. We find that conversion may have played a dominant role in shaping haplotype patterns over short stretches of DNA, whereas reciprocal recombination may have played a greater role over longer stretches of DNA. However, in order to draw firm conclusions more independent data are necessary.

Comparative sequence analysis of the MECP2-locus in human and mouse reveals new transcribed regions.

Comparative sequence analysis facilitates the identification of evolutionarily conserved regions, that is, gene-regulatory elements, which can not be detected by analyzing one species only. Sequencing of a 152-kb region on human Chromosome (Chr) Xq28 and of the synthenic 123 kb on mouse Chr XC identified the MECP2/Mecp2 locus, which is flanked by the gene coding for Interleukin-1 receptor associated kinase (IRAK/Il1rak) and the red opsin gene (RCP/Rsvp). By comparative sequence analysis, we identified a previously unknown, non-coding 5' exon embedded in a CpG island associated with MECP2/Mecp2. Thus, the MECP2/Mecp2 gene is comprised of four exons instead of three. Furthermore, sequence comparison 3' to the previously reported polyadenylation signal revealed a highly conserved region of 8.5 kb terminating in an alternative polyadenylation signal. Northern blot analysis verified the existence of two main transcripts of 1.9 kb and approximately 10 kb, respectively. Both transcripts exhibit tissue-specific expression patterns and have almost identical short half-lifes. The approximately 10-kb transcript corresponds to a giant 3' UTR contained in the fourth exon of MECP2. The long 3' UTR and the newly identified first intron of MECP2/Mecp2 are highly conserved in human and mouse. Furthermore, the human MECP2 locus is heterogeneous with respect to its DNA composition. We postulate that it represents a boundary between two H3 isochores that has not been observed previously.

Genome sequence comparisons: hurdles in the fast lane to functional genomics.

An important computational technique for extracting the wealth of information hidden in human genomic sequence data is to compare the sequence with that from the corresponding region of the mouse genome, looking for segments that are conserved over evolutionary time. Moreover, the approach generalises to comparison of sequences from any two related species. The underlying rationale (which is abundantly confirmed by observation) is that a random mutation in a functional region is usually deleterious to the organism, and hence unlikely to become fixed in the population, whereas mutations in a non-functional region are free to accumulate over time. The potential value of this approach is so attractive that the public and private projects to sequence the human genome are now turning to sequencing the mouse, and you will soon be able to compare the human and mouse sequences of your favourite genomic region. We are currently witnessing an explosion of computer tools for comparative analysis of two genomic sequences. Here the capabilities of two new network servers for comparing genomic sequences from any pair of closely related species are sketched. The Syntenic Gene Prediction Program SGP-I utilises sequence comparisons to enhance the ability to locate protein coding segments in genomic data. PipMaker attempts to determine all conserved genomic regions, regardless of their function.

Post-processing long pairwise alignments.

MOTIVATION: The local alignment problem for two sequences requires determining similar regions, one from each sequence, and aligning those regions. For alignments computed by dynamic programming, current approaches for selecting similar regions may have potential flaws. For instance, the criterion of Smith and Waterman can lead to inclusion of an arbitrarily poor internal segment. Other approaches can generate an alignment scoring less than some of its internal segments. RESULTS: We develop an algorithm that decomposes a long alignment into sub-alignments that avoid these potential imperfections. Our algorithm runs in time proportional to the original alignment's length. Practical applications to alignments of genomic DNA sequences are described.

Genomic organization of a 225-kb region in Xq28 containing the gene for X-linked myotubular myopathy (MTM1) and a related gene (MTMR1).

MTM1 is responsible for X-linked recessive myotubular myopathy, which is a congenital muscle disorder linked to Xq28. MTM1 is highly conserved from yeast to humans. A number of related genes also exist. The MTM1 gene family contains a consensus sequence consisting of the active enzyme site of protein tyrosine phosphatases (PTPs), suggesting that they belong to a new family of PTPs. Database searches revealed homology of myotubularin and all related peptides to the cisplatin resistance-associated alpha protein, which implicates an as yet unknown function. In addition, homology to the Sbf1 protein (SET binding factor 1), involved in the oncogenic transformation of fibroblasts and differentiation of myoblasts, was also evident. We describe 225 kb of genomic sequence containing MTM1 and the related gene, MTMR1, which lies 20 kb distal to MTM1. Although there is only moderate conservation of the exons, the striking similarity in the gene structures indicates that these two genes arose by duplication. Calculations suggest that this event occurred early in evolution long before separation of the human and mouse lineages. So far, mutations have been identified in the coding sequence of only 65% of the patients analyzed, indicating that the remaining mutations may lie in noncoding regions of MTM1 or possibly in MTMR1. Knowledge of the genomic sequence will facilitate mutation analyses of the coding and noncoding sequences of MTM1 and MTMR1.

Genetic hitch-hiking in a subdivided population.

The problem of genetic hitch-hiking in a geographically subdivided population is analysed under the assumption that migration rates among populations are relatively small compared with the selection coefficient for a newly arising advantageous allele. The approximate method used in the paper is valid when the number of emigrants per generation (Nm) is less than one. The approximate analysis shows that hitch-hiking can result in substantial differences among populations in the frequencies of neutral alleles closely linked to the advantageous allele. Thus, in cases for which genetic hitch-hiking is thought to be responsible for low levels of genetic variability in regions of the genome with restricted crossing over, it might be possible to find confirmatory evidence for that hypothesis by finding unusual patterns of geographic differentiation in the same regions of the genome.

The effect of selective sweeps on the variance of the allele distribution of a linked multiallele locus: hitchhiking of microsatellites.

Microsatellite variation and the mechanisms which are responsible for this variation have received much attention in the last few years. Most theoretical studies of microsatellite allele distributions, however, did not incorporate the evolutionary dynamics of linked sites. The dynamics is usually modeled by invoking a special mutation mechanism such as stepwise mutation, which leads to a stepwise increase or decrease of the number of motif repeats on the occasion of mutation. It is shown here that selection at a locus, which itself is not subject to mutation, but which is adjacent to a microsatellite locus has an influence on statistics of the microsatellite allele distribution, provided that mutation rates are low to intermediate, when compared to 1/t1, the inverse of the time to fixation of a linked favorable substitution. If mutation rates are high, as for example in humans, a selective effect upon the microsatellite locus, such as hitchhiking, will quickly be obscured by mutations. In particular, in the latter case, the model shows that no correlation is to be expected between recombination rates and variability of microsatellites--such as had been predicted and experimentally demonstrated for nucleotide variability and recombination rates in Drosophila. The present model is a generalization of the two locus two allele hitchhiking model which had been studied by Stephan and co-workers.

Epistatic selection in a multi-locus Levene model and implications for linkage disequilibrium.

We analyze a multiple-locus extension of the Levene (1953) model of population subdivision. We show that stable or quasistable linkage disequilibrium between two selected loci can be maintained even with free recombination, provided that there is a strong enough epistatic interaction. We then consider the dynamics of a third neutral locus and show that its approach to linkage equilibrium depends on the recombination rates and the selection intensities. There is an embedding or hitchhiking effect that extends the time during which a neutral locus which is closely linked to one of the selected loci remains in disequilibrium with both selected loci. Therefore, strong disequilibrium between two loci does not necessarily indicate that those loci are themselves selected, but it does indicate that there is strong selection acting at least on nearby loci. This property implies a warning that screening for linkage disequilibrium as a tool to identify functionally important sites in a genome can be misleading.

Error propagation in reproduction of diploid organisms. A case study on single peaked landscapes.

Two versions of the diploid selection mutation equation as adapted to sequence space are studied. Focussing on diploid generalizations of the well-established single peaked landscape, quantitative effects of dominance on error thresholds in infinite populations are found, as well as unexpected qualitative features like multiple equilibria. Analogues of these phenomena are also recovered in stochastic versions for finite populations.

Analysis of a genetic hitchhiking model, and its application to DNA polymorphism data from Drosophila melanogaster.

Begun and Aquadro have demonstrated that levels of nucleotide variation correlate with recombination rate among 20 gene regions from across the genome of Drosophila melanogaster. It has been suggested that this correlation results from genetic hitchhiking associated with the fixation of strongly selected mutants. The hitchhiking process can be described as a series of two-step events. The first step consists of a strongly selected substitution wiping out linked variation in a population; this is followed by a recovery period in which polymorphism can build up via neutral mutations and random genetic drift. Genetic hitchhiking has previously been modeled as a steady-state process driven by recurring selected substitutions. We show here that the characteristic parameter of this steady-state model is alpha v, the product of selection intensity (alpha = 2Ns) and the frequency of beneficial mutations v (where N is population size and s is the selective advantage of the favored allele). We also demonstrate that the steady-state model describes the hitchhiking process adequately, unless the recombination rate is very low. To estimate alpha v, we use the data of DNA sequence variation from 17 D. melanogaster loci from regions of intermediate to high recombination rates. We find that alpha v is likely to be > 1.3 x 10(-8). Additional data are needed to estimate this parameter more precisely. The estimation of alpha v is important, as this parameter determines the shape of the frequency distribution of strongly selected substitutions.