The phylogenetic likelihood library.

We introduce the Phylogenetic Likelihood Library (PLL), a highly optimized application programming interface for developing likelihood-based phylogenetic inference and postanalysis software. The PLL implements appropriate data structures and functions that allow users to quickly implement common, error-prone, and labor-intensive tasks, such as likelihood calculations, model parameter as well as branch length optimization, and tree space exploration. The highly optimized and parallelized implementation of the phylogenetic likelihood function and a thorough documentation provide a framework for rapid development of scalable parallel phylogenetic software. By example of two likelihood-based phylogenetic codes we show that the PLL improves the sequential performance of current software by a factor of 2-10 while requiring only 1 month of programming time for integration. We show that, when numerical scaling for preventing floating point underflow is enabled, the double precision likelihood calculations in the PLL are up to 1.9 times faster than those in BEAGLE. On an empirical DNA dataset with 2000 taxa the AVX version of PLL is 4 times faster than BEAGLE (scaling enabled and required). The PLL is available at http://www.libpll.org under the GNU General Public License (GPL).

The genetical archaeology of the human genome.

Palaentology and archaeology are disciplines that traditionally deal with the reconstruction of human origins and history. Recently, however, molecular genetics has come to make increasing contributions to this area. In particular, several data sets indicate that variation of the human gene pool originated in Africa within the last 200,000 years. Furthermore, the study of DNA sequences allows the detection of expansions in population size. Here we briefly summarize and exemplify how DNA sequences can be used to reconstruct the history of populations.

DNA sequence variation in a non-coding region of low recombination on the human X chromosome.

DNA sequence variation has become a major source of insight regarding the origin and history of our species as well as an important tool for the identification of allelic variants associated with disease. Comparative sequencing of DNA has to date focused mainly on mitochondrial (mt) DNA, which due to its apparent lack of recombination and high evolutionary rate lends itself well to the study of human evolution. These advantages also entail limitations. For example, the high mutation rate of mtDNA results in multiple substitutions that make phylogenetic analysis difficult and, because mtDNA is maternally inherited, it reflects only the history of females. For the history of males, the non-recombining part of the paternally inherited Y chromosome can be studied. The extent of variation on the Y chromosome is so low that variation at particular sites known to be polymorphic rather than entire sequences are typically determined. It is currently unclear how some forms of analysis (such as the coalescent) should be applied to such data. Furthermore, the lack of recombination means that selection at any locus affects all 59 Mb of DNA. To gauge the extent and pattern of point substitutional variation in non-coding parts of the human genome, we have sequenced 10 kb of non-coding DNA in a region of low recombination at Xq13.3. Analysis of this sequence in 69 individuals representing all major linguistic groups reveals the highest overall diversity in Africa, whereas deep divergences also exist in Asia. The time elapsed since the most recent common ancestor (MRCA) is 535,000+/-119,000 years. We expect this type of nuclear locus to provide more answers about the genetic origin and history of humans.

Genomic SELEX for Hfq-binding RNAs identifies genomic aptamers predominantly in antisense transcripts.

An unexpectedly high number of regulatory RNAs have been recently discovered that fine-tune the function of genes at all levels of expression. We employed Genomic SELEX, a method to identify protein-binding RNAs encoded in the genome, to search for further regulatory RNAs in Escherichia coli. We used the global regulator protein Hfq as bait, because it can interact with a large number of RNAs, promoting their interaction. The enriched SELEX pool was subjected to deep sequencing, and 8865 sequences were mapped to the E. coli genome. These short sequences represent genomic Hfq-aptamers and are part of potential regulatory elements within RNA molecules. The motif 5'-AAYAAYAA-3' was enriched in the selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the antisense strand of protein coding genes than on the sense strand. They were enriched opposite to translation start sites or opposite to intervening sequences between ORFs in operons. These results expand the repertoire of Hfq targets and also suggest that Hfq might regulate the expression of a large number of genes via interaction with cis-antisense RNAs.

How old is the genetic code? Statistical geometry of tRNA provides an answer.

The age of the molecular organization of life as expressed in the genetic code can be estimated from experimental data. Comparative sequence analysis of transfer RNA by the method of statistical geometry in sequence space suggests that about one-third of the present transfer RNA sequence divergence was present at the urkingdom level about the time when archaebacteria separated from eubacteria. It is concluded that the genetic code is not older than, but almost as old as our planet. While this result may not be unexpected, it was not clear until now that interpretable data exist that permit inferences about such early stages of life as the establishment of the genetic code.

Molecular signature of CD34(+) hematopoietic stem and progenitor cells of patients with CML in chronic phase.

In this study, we provide a molecular signature of highly enriched CD34+ cells from bone marrow of untreated patients with chronic myelogenous leukemia (CML) in chronic phase in comparison with normal CD34+ cells using microarrays covering 8746 genes. Expression data reflected several BCR-ABL-induced effects in primary CML progenitors, such as transcriptional activation of the classical mitogen-activated protein kinase pathway and the phosphoinositide-3 kinase/AKT pathway as well as downregulation of the proapoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include upregulation of genes involved in the transforming growth factorbeta pathway, fetal hemoglobin genes, leptin receptor, sorcin, tissue inhibitor of metalloproteinase 1, the neuroepithelial cell transforming gene 1 and downregulation of selenoprotein P. Additionally, genes associated with early hematopoietic stem cells (HSC) and leukemogenesis such as HoxA9 and MEIS1 were transcriptionally activated. Differential expression of differentiation-associated genes suggested an altered composition of the CD34+ cell population in CML. This was confirmed by subset analyses of chronic phase CML CD34+ cells showing an increase of the proportion of megakaryocyte-erythroid progenitors, whereas the proportion of HSC and granulocyte-macrophage progenitors was decreased in CML. In conclusion, our results give novel insights into the biology of CML and could provide the basis for identification of new therapeutic targets.

DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA.

We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C-->T and G-->A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, 'jumping PCR', i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than approximately 0.1% even under the unlikely scenario where each amplification starts from a single template molecule.

A protein kinase from Dictyostelium discoideum with an unusual acidic repeat domain.

DdKinX codes for 1093 amino acids which are organized in four regions: the N-terminal catalytic domain, a region containing 30% acidic amino acids, tandem repeats of the motif VKVEEPVEE and the C-terminus. Identity with other protein kinases is 25 to 30%. Descendent trees show that DdKinX does not belong to any of the known kinase branches.

Inference of population history using a likelihood approach.

We introduce an approach to revealing the likelihood of different population histories that utilizes an explicit model of sequence evolution for the DNA segment under study. Based on a phylogenetic tree reconstruction method we show that a Tamura-Nei model with heterogeneous mutation rates is a fair description of the evolutionary process of the hypervariable region I of the mitochondrial DNA from humans. Assuming this complex model still allows the estimation of population history parameters, we suggest a likelihood approach to conducting statistical inference within a class of expansion models. More precisely, the likelihood of the data is based on the mean pairwise differences between DNA sequences and the number of variable sites in a sample. The use of likelihood ratios enables comparison of different hypotheses about population history, such as constant population size during the past or an increase or decrease of population size starting at some point back in time. This method was applied to show that the population of the Basques has expanded, whereas that of the Biaka pygmies is most likely decreasing. The Nuu-Chah-Nulth data are consistent with a model of constant population.

Pattern of nucleotide substitution and rate heterogeneity in the hypervariable regions I and II of human mtDNA.

This study provides a comprehensive survey of the complex pattern of nucleotide substitution in the control region of human mtDNA, which is of central importance to the studies of human evolution. A total of 1229 different hypervariable region I (HVRI) and 385 different hypervariable region II (HVRII) sequences were analyzed using a complex substitution model. Moreover, we suggest a new method to assign relative rates to each site in the sequence. Estimates are based on maximum-likelihood methods applied to randomly selected subsets of sequences. Our results indicate that the rate of substitution in HVRI is approximately twice as high as in HVRII and that this difference is mainly due to a higher frequency of pyrimidine transitions in HVRI. However, rate heterogeneity is more pronounced in HVRII.

The marsupial mitochondrial genome and the evolution of placental mammals.

The entire nucleotide sequence of the mitochondrial genome of the American opossum, Didelphis virginiana, was determined. Two major features distinguish this genome from those of other mammals. First, five tRNA genes around the origin of light strand replication are rearranged. Second, the anticodon of tRNA(Asp) is posttranscriptionally changed by an RNA editing process such that its coding capacity is altered. When the complete protein-coding region of the mitochondrial genome is used as an outgroup for placental mammals it can be shown that rodents represent an earlier branch among placental mammals than primates and artiodactyls and that artiodactyls share a common ancestor with carnivores. The overall rates of evolution of most of the mitochondrial genome of placentals are clock-like. Furthermore, the data indicate that the lineages leading to the mouse and rat may have diverged from each other as much as 35 million years ago.

Modeling the polymerase chain reaction.

We introduce a mathematical model to treat the polymerase chain reaction (PCR), where we regard the accumulation of new molecules during a PCR cycle as a randomly bifurcating tree. This model enables us to compute an approximate formula for the distribution of the number of replications that have occurred between a pair of molecules, which depends on the efficiency lambda of the reaction, the number N0 of template molecules at the beginning of the PCR and the number c of PCR cycles. The reliability of the approximation is tested by computer simulations. Finally, to model the effect of the intrinsic error rate of the polymerase, we superimpose a substitution process on the tree. The resulting closed formula for the distribution of pairwise differences of sequences as a function of error rate mu and efficiency lambda can be used to estimate the error rate, if lambda is known.

Evolution of DNA or amino acid sequences with dependent sites.

A framework is outlined to study the evolution of DNA or amino acid sequences, if sequence sites do not evolve independently. The units of evolution are nonoverlapping subsequences of length l. Each subsequence evolves independently of the others, but within a subsequence the sequences show a Markov order one dependency. We describe an algorithm to mimic the evolution of such sequences. The influence of dependencies between sites on distance estimates and the reliability of tree reconstruction methods is investigated. We show that an inappropriate model of sequence evolution in the tree reconstruction process will lead to a nonempty Felsenstein zone. Finally, we describe a method to infer l from sequence data. Examples from the evolution of DNA sequences as well as from amino acids are given.

Towards integration of multiple alignment and phylogenetic tree construction.

A central problem in the study of molecular evolution is the reconstruction of the history of a set of biological sequences in the form of a phylogenetic tree. One step in calculating this tree is the computation of a multiple alignment. Most existing approaches treat the two problems of multiple alignment and tree construction as separate while in fact they influence each other. Based on three-way alignments of pre-aligned groups of sequences we adapt a commonly used tree construction procedure to produce both tree and multiple alignment simultaneously. In contrast to existing iterative algorithms the new method can change alignments made early in the course of the computation at a later stage. A sufficient criterion to prevent the introduction of edges with negative length reduces the number of three-way alignments that need to be computed. Applications of the new approach to the alignment of protein and of nucleic acid sequences are presented.

Phylogenetic analysis of rubella virus including new genotype I isolates.

Infection during the first trimester of gestation with rubella virus (RV) is highly teratogenic. Embryopathy is a frequent outcome of the primary natural infection with wild type RV during pregnancy while accidental immunisation with life attenuated vaccine has apparently little or no adverse effect. Although the nucleic acid sequence of RV is exceptionally stable, differences between the vaccine and wild type viruses could play a role in the pathogenesis of intrauterine RV infection. Phylogenetic analysis of eight complete sequences (including two new isolates described in this paper, three vaccine strains, three cell culture adapted wild type viruses) confirms a striking low divergence of the RV genome. A variable region (amino acid residues 697-800) within the gene coding for the nonstructural protein NSP1 was defined. Phylogenetic analysis revealed a strong positive selection in this region. Multiple passages in vivo or in vitro did not account for this variability. As the function of the variable region has not yet been elucidated, reasons for and significance of positive selection are still speculative. It is conceivable that the variable region in NSP1 contributes to the molecular basis of RV embryopathy and other complications of postnatal RV infection.

Mitochondrial DNA sequencing of shed hairs and saliva on robbery caps: sensitivity and matching probabilities.

Sequencing of mitochondrial DNA (mtDNA) has been used for human identification based on teeth and skeletal remains. Here, we describe an amplification system for the mtDNA control region (D-loop) suited for the analysis of shed hair, which constitutes the most common biological evidence material in forensic investigations. The success rate was over 90% when applied to evidence materials such as shed hair, saliva stains and saliva on stamps. The analysis of evidence materials collected from three similar robberies revealed the presence of mtDNA sequences identical to those of the suspects in the three crimes. The use of mtDNA control region sequences for individual identification was evaluated. The probability of identity by chance for the mtDNA types of the suspects in the robberies was found to vary between Pr = 0.017 - < 0.0017, depending on the reference population used, emphasizing the need for large population databases to obtain the appropriate estimate.

Network models for sequence evolution.

We introduce a general class of models for sequence evolution that includes network phylogenies. Networks, a generalization of strictly tree-like phylogenies, are proposed to model situations where multiple lineages contribute to the observed sequences. An algorithm to compute the probability distribution of binary character-state configurations is presented and statistical inference for this model is developed in a likelihood framework. A stepwise procedure based on likelihood ratios is used to explore the space of models. Starting with a star phylogeny, new splits (nontrivial bipartitions of the sequence set) are successively added to the model until no significant change in the likelihood is observed. A novel feature of our approach is that the new splits are not necessarily constrained to be consistent with a treelike mode of evolution. The fraction of invariable sites is estimated by maximum likelihood simultaneously with other model parameters and is essential to obtain a good fit to the data. The effect of finite sequence length on the inference methods is discussed. Finally, we provide an illustrative example using aligned VP1 genes from the foot and mouth disease viruses (FMDV). The different serotypes of the FMDV exhibit a range of treelike and network evolutionary relationships.