Genomic GC Research Articles

Predictive role of mitochondrial genome in the stress resistance of insects and nematodes

Certain insects (e.g., moths and butterflies; order Lepidoptera) and nematodes are considered as excellent experimental models to study the cellular stress signaling mechanisms since these organisms are far more stress-resistant as compared to mammalian system. Multiple factors have been implicated in this unusual response, including the oxidative stress response mechanisms. Radiation or chemical-induced mitochondrial oxidative stress occurs through damage caused to the components of electron transport chain (ETC) leading to leakage of electrons and generation of superoxide radicals. This may be countered through quick replacement of damaged mitochondrial proteins by upregulated expression. Since the ETC comprises of various proteins coded by mitochondrial DNA, variation in the composition, expressivity and regulation of mitochondrial genome could greatly influence mitochondrial role under oxidative stress conditions. Therefore, we carried out in silico analysis of mitochondrial DNA in these organisms and compared it with that of the stress-sensitive humans/mammals. Parameters such as mitochondrial genome organization, codon bias, gene expressivity and GC(3) content were studied. Gene arrangement and Shine-Dalgarno (SD) sequence patterns indicating translational regulation were distinct in insect and nematodes as compared to humans. A higher codon bias (ENC≫35) and lower GC(3) content (≫0.20) were observed in mitochondrial genes of insect and nematodes as compared to humans (ENC>42; GC3>0.20), coupled with low codon adaptation index among insects. These features indeed favour higher expressivity of mitochondrial proteins and might help maintain the mitochondrial physiology under stress conditions. Therefore, our study indicates that mitochondrial genome organization may influence stress-resistance of insects and nematodes.

Sensitivity of Noninvasive Prenatal Detection of Fetal Aneuploidy from Maternal Plasma Using Shotgun Sequencing Is Limited Only by Counting Statistics

We recently demonstrated noninvasive detection of fetal aneuploidy by shotgun sequencing cell-free DNA in maternal plasma using next-generation high throughput sequencer. However, GC bias introduced by the sequencer placed a practical limit on the sensitivity of aneuploidy detection. In this study, we describe a method to computationally remove GC bias in short read sequencing data by applying weight to each sequenced read based on local genomic GC content. We show that sensitivity is limited only by counting statistics and that sensitivity can be increased to arbitrary precision in sample containing arbitrarily small fraction of fetal DNA simply by sequencing more DNA molecules. High throughput shotgun sequencing of maternal plasma DNA should therefore enable noninvasive diagnosis of any type of fetal aneuploidy.

Codon Usage Biases in Alzheimers Disease and Other Neurodegenerative Diseases

Establishing codon usage biases are crucial for understanding the etiology of central nervous system neurodegenerative diseases (CNSNDD) especially Alzheimer's disease (AD) as well as genetic factors. G and C ending codons are strongly biased in the coding sequences of these proteins as a result of genomic GC composition constraints. On the other hand, codons that identified as translationally optimal in the major trend all end in C or G, suggesting translational selection should also be taken into consideration additional to compositional constraints. Furthermore, this investigation reveals that three common codons, CGC (Arg), AGC (Ser), and GGC (Gly), are also critical in affecting codon usage bias. They not only can offer an insight into the codon usage bias of AD and its mechanism, but also may help in the possible cures for these diseases.

Abstract B9: Germline conservation of hypermutable codons in gatekeeper tumor suppressor genes implicates pro-carcinogenic CpG sites in vivo as anti-teratogenic ‘shock absorbers’ in utero

Abstract Purpose: Methylated CpG sites are readily mutated, yet are strongly conserved in tumor suppressor genes. Uniquely, the arginine-encoding CGA codon may undergo methylation-dependent point mutation to a stop codon (TGA), whereas the arginine-encoding AGA codon is stable. To clarify the relationship between germline codon mutability and somatic carcinogenesis, we compared patterns of codon usage in tumor suppressor gene subsets known to differ in function and evolutionary stability. Procedures: Human tumor suppressor genes were classified as either pro-apoptotic (gatekeepers; high-essentiality, low Ka/Ks) or repair-type (caretakers; low-essentiality, high Ka/Ks). Reference normal and tumor gene sequence databases were analysed in terms of nucleotide composition (GC%), intragenic CpG sites, and CGA/AGA codon patterns. Phylogenetic analyses of relevant codon frequencies across 24 species from the UCSC database were performed as a control. Results: Species-specific stop codon frequency varies with genomic GC content in a methylation-regulated manner, whereas CGA frequency is GC-independent. Whole-genome frequencies of CGA and TGA codons are positively correlated (p = 0.013). CGA mutation in tumors is asymmetric (TGA >> CAA; p < 0.001), with such mutations 3-fold more likely to be nonsense-type in gatekeepers than in caretakers. Non-random CGA clustering is demonstrable in canonical gatekeepers such as NF1, Rb and TP53 (p < 0.02). Conclusion: Human pro-apoptotic suppressor genes are selectively enriched for the presence of nonsense-prone CpG-containing codons involved in somatic adult-onset tumor progression. We propose that this germline vulnerability is conserved as part of a methylation-dependent embryonic response to environmental teratogens which otherwise cause excessive apoptosis, somatic malformations, and reduced fitness. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B9

Metabolic rate and genomic GC. What we can learn from teleost fish

Teleosts are a highly diverse group of animals occupying all kind of aquatic environment. Data on routine mass specific metabolic rate were re-examined correcting them for the Boltzmann's factor. Teleostean fish were grouped in five broad groups, corresponding to major environmental classifications: polar, temperate, sub-tropical, tropical and deep-water. The specific routine metabolic rate, temperature-corrected using the Boltzmann's factor (MR), and the average base composition of genomes (GC%) were calculated in each group. Fish of the polar habitat showed the highest MR. Temperate fish displayed a significantly higher MR than tropical fish, which had the lowest average value. These results were apparently in agreement with the cold adaptation hypothesis. In contrast with this hypothesis, however, the MR of fish living in deep-water environment turned out to be not significantly different from that of fish living in tropical habitats. Most probably, the amount of oxygen dissolved in the water directly affects MR adaptation.Regarding the different habitats, the genomic GC levels showed a decreasing trend similar to that of MR. Indeed, both polar and temperate fish showed a GC level significantly higher than that of both sub-tropical and tropical fish.Plotting the genomic GC levels versus the MR a significant positive correlation was found, supporting the hypothesis that metabolic rate can explain not only the compositional transition mode (e.g. amphibian/mammals), but also the compositional shifting mode (e.g. fish/fish) of evolution observed for vertebrate genomes.

Genomic adaptation of prokaryotic organisms at high temperature

One of the central issues of evolutionary genomics is to find out the adaptive strategies of microorganisms to stabilize nucleic acid molecules under high temperature. Thermal adaptation hypothesis gives a link between G+C content and growth temperature if there is a considerable variation of guanine and cytosine content between species. However, there has been a long-standing debate regarding the correlations between genomic GC content and optimal growth temperature (Topt). We urged that adaptation to growth at high temperature requires a coordinated set of evolutionary changes affecting: (i) nucleic acid thermostability and (ii) stability of codon-anticodon interactions. Moreover, in Bacillaceae family we have demonstrated that a higher genomic GC level do not have any role in stabilizing mRNA secondary structure at high growth temperature. Comparative analysis between homologous sequences of thermophilic Thermus thermophilus and mesophilic Deinococcus radiodurans suggests that increased levels of GC contents in the coding sequence corresponding to strand structure of Thermus thermophilus genes have stabilizing effect on the mRNA secondary structure, whereas increased levels of GC contents in coding sequences corresponding to aperiodic structure have destabilizing effect on the mRNA secondary structure. In this perspective, a critical review of thermal adaptation hypothesis is further advocated.

Higher tRNA diversity in thermophilic bacteria: A possible adaptation to growth at high temperature

We have done a comparative study of tRNA diversity and total tRNA genes among different strains of bacteria with respect to the optimum growth temperature of the cells. Our observation suggests that higher tRNA diversity usually occurs in thermophiles in comparison to non-thermophiles. Among psychrophiles total tRNA was observed to be more than two-fold higher than in the non-psychrophiles. Though tRNA diversity and total tRNA have recently been shown to be affected by an organism's genomic GC% and growth rate, this work is the first report on growth temperature affecting these features in bacteria. This work extends the list of molecular features undergoing adaptation due to growth temperature and supports the view that growth temperature acts as a selecting factor at the molecular level during evolution.

A Universal Trend of Reduced mRNA Stability near the Translation-Initiation Site in Prokaryotes and Eukaryotes

Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA.

Analysis of intra-genomic GC content homogeneity within prokaryotes

BackgroundBacterial genomes possess varying GC content (total guanines (Gs) and cytosines (Cs) per total of the four bases within the genome) but within a given genome, GC content can vary locally along the chromosome, with some regions significantly more or less GC rich than on average. We have examined how the GC content varies within microbial genomes to assess whether this property can be associated with certain biological functions related to the organism's environment and phylogeny. We utilize a new quantity GCVAR, the intra-genomic GC content variability with respect to the average GC content of the total genome. A low GCVAR indicates intra-genomic GC homogeneity and high GCVAR heterogeneity.ResultsThe regression analyses indicated that GCVAR was significantly associated with domain (i.e. archaea or bacteria), phylum, and oxygen requirement. GCVAR was significantly higher among anaerobes than both aerobic and facultative microbes. Although an association has previously been found between mean genomic GC content and oxygen requirement, our analysis suggests that no such association exits when phylogenetic bias is accounted for. A significant association between GCVAR and mean GC content was also found but appears to be non-linear and varies greatly among phyla.ConclusionsOur findings show that GCVAR is linked with oxygen requirement, while mean genomic GC content is not. We therefore suggest that GCVAR should be used as a complement to mean GC content.

Examination of Genome Homogeneity in Prokaryotes Using Genomic Signatures

BackgroundDNA word frequencies, normalized for genomic AT content, are remarkably stable within prokaryotic genomes and are therefore said to reflect a “genomic signature.” The genomic signatures can be used to phylogenetically classify organisms from arbitrary sampled DNA. Genomic signatures can also be used to search for horizontally transferred DNA or DNA regions subjected to special selection forces. Thus, the stability of the genomic signature can be used as a measure of genomic homogeneity. The factors associated with the stability of the genomic signatures are not known, and this motivated us to investigate further. We analyzed the intra-genomic variance of genomic signatures based on AT content normalization (0th order Markov model) as well as genomic signatures normalized by smaller DNA words (1st and 2nd order Markov models) for 636 sequenced prokaryotic genomes. Regression models were fitted, with intra-genomic signature variance as the response variable, to a set of factors representing genomic properties such as genomic AT content, genome size, habitat, phylum, oxygen requirement, optimal growth temperature and oligonucleotide usage variance (OUV, a measure of oligonucleotide usage bias), measured as the variance between genomic tetranucleotide frequencies and Markov chain approximated tetranucleotide frequencies, as predictors.Principal FindingsRegression analysis revealed that OUV was the most important factor (p<0.001) determining intra-genomic homogeneity as measured using genomic signatures. This means that the less random the oligonucleotide usage is in the sense of higher OUV, the more homogeneous the genome is in terms of the genomic signature. The other factors influencing variance in the genomic signature (p<0.001) were genomic AT content, phylum and oxygen requirement.ConclusionsGenomic homogeneity in prokaryotes is intimately linked to genomic GC content, oligonucleotide usage bias (OUV) and aerobiosis, while oligonucleotide usage bias (OUV) is associated with genomic GC content, aerobiosis and habitat.

MITOCHONDRIAL DNA IN THE OOGAMOCHLAMYS CLADE (CHLOROPHYCEAE): HIGH GC CONTENT AND UNIQUE GENOME ARCHITECTURE FOR GREEN ALGAE(1).

Most mitochondrial genomes in the green algal phylum Chlorophyta are AT-rich, circular-mapping DNA molecules. However, mitochondrial genomes from the Reinhardtii clade of the Chlorophyceae lineage are linear and sometimes fragmented into subgenomic forms. Moreover, Polytomella capuana, from the Reinhardtii clade, has an elevated GC content (57.2%). In the present study, we examined mitochondrial genome conformation and GC bias in the Oogamochlamys clade of the Chlorophyceae, which phylogenetic data suggest is closely related to the Reinhardtii clade. Total DNA from selected Oogamochlamys taxa, including four Lobochlamys culleus (H. Ettl) Pröschold, B. Marin, U. G. Schlöss. et Melkonian strains, Lobochlamys segnis (H. Ettl) Pröschold, B. Marin, U. G. Schlöss. et Melkonian, and Oogamochlamys gigantea (O. Dill) Pröschold, B. Marin, U. G. Schlöss. et Melkonian, was subjected to Southern blot analyses with cob and cox1 probes, and the results suggest that the mitochondrial genome of these taxa is represented by multiple-sized linear DNA fragments with overlapping homologies. On the basis of these data, we propose that linear mitochondrial DNA with a propensity to become fragmented arose in an ancestor common to the Reinhardtii and Oogamochlamys clades or even earlier in the evolutionary history of the Chlorophyceae. Analyses of partial cob and cox1 sequences from these Oogamochlamys taxa revealed an unusually high GC content (49.9%-65.1%) and provided evidence for the accumulation of cob and cox1 pseudogenes and truncated sequences in the mitochondrial genome of all L. culleus strains examined.

GC Content-Based Pan-Pox Universal PCR Assays for Poxvirus Detection

Chordopoxviruses of the subfamily Chordopoxvirinae, family Poxviridae, infect vertebrates and consist of at least eight genera with broad host ranges. For most chordopoxviruses, the number of viral genes and their relative order are highly conserved in the central region. The GC content of chordopoxvirus genomes, however, evolved into two distinct types: those with genome GC content of more than 60% and those with a content of less than 40% GC. Two standard PCR assays were developed to identify chordopoxviruses based on whether the target virus has a low or high GC content. In design of the assays, the genus Avipoxvirus, which encodes major rearrangements of gene clusters, was excluded. These pan-pox assays amplify DNA from more than 150 different isolates and strains, including from primary clinical materials, from all seven targeted genera of chordopoxviruses and four unclassified new poxvirus species. The pan-pox assays represent an important advance for the screening and diagnosis of human and animal poxvirus infections, and the technology used is accessible to many laboratories worldwide.

Variable correlation of genome GC% with transfer RNA number as well as with transfer RNA diversity among bacterial groups: α-Proteobacteria and Tenericutes exhibit strong positive correlation

Mainly two selective forces are thought to affect transfer RNA (tRNA) gene numbers in cells: firstly, transfer RNA genes have coevolved with codon usage to optimize the translation rate in organisms; and secondly, tRNA gene number is positively correlated with growth rate of organisms. Since a codon ending with 'G' is recognized with lower efficiency than the synonymous codon ending with 'A' by a tRNA, organisms with high genome GC% are expected to possess tRNA molecules with 'C' at the first anticodon position to decode efficiently the former codon. This is in concordance with the observation of higher tRNA diversity in several bacteria with higher genome GC%. In addition to this, tRNA gene redundancy has been reported to be correlated with genome size. In this paper we have carried out a comprehensive correlation analysis of tRNA gene diversity, total tRNA number, genome GC% and genome size, in 626 bacterial strains (belonging to 433 species). A common positive trend of correlation in bacteria of GC% with tRNA diversity, of GC% with genome size and of genome size with total tRNA number, is observed, even within individual bacterial groups. Comparison of organisms' growth rate data with their genome GC% indicated that organisms with higher growth rate are most likely with <50% GC. This work extends the list of features in organisms that relate to genome GC%.

General Rules for Optimal Codon Choice

Different synonymous codons are favored by natural selection for translation efficiency and accuracy in different organisms. The rules governing the identities of favored codons in different organisms remain obscure. In fact, it is not known whether such rules exist or whether favored codons are chosen randomly in evolution in a process akin to a series of frozen accidents. Here, we study this question by identifying for the first time the favored codons in 675 bacteria, 52 archea, and 10 fungi. We use a number of tests to show that the identified codons are indeed likely to be favored and find that across all studied organisms the identity of favored codons tracks the GC content of the genomes. Once the effect of the genomic GC content on selectively favored codon choice is taken into account, additional universal amino acid specific rules governing the identity of favored codons become apparent. Our results provide for the first time a clear set of rules governing the evolution of selectively favored codon usage. Based on these results, we describe a putative scenario for how evolutionary shifts in the identity of selectively favored codons can occur without even temporary weakening of natural selection for codon bias.

Occurrence, divergence and evolution of intrinsic terminators across Eubacteria

In Escherichia coli, the canonical intrinsic terminator of transcription includes a palindrome followed by a U-trail on the transcript. The apparent underrepresentation of such terminators in eubacterial genomes led us to develop a rapid and accurate algorithm, GeSTer, to predict putative intrinsic terminators. Now, we have analyzed 378 genome sequences with an improved version of GeSTer. Our results indicate that the canonical E. coli type terminators are not overwhelmingly abundant in eubacteria. The atypical structures, having stem-loop structures but lacking ‘U’ trail, occur downstream of genes in all the analyzed genomes but different phyla show conserved preference for different types of terminators. This propensity correlates with genomic GC content and presence of the factor, Rho. 60–70% of identified terminators in all the genomes show “optimized” stem-length and Δ G. These results provide evidence that eubacteria extensively rely on the mechanism of intrinsic termination, with a considerable divergence in their structure, positioning and prevalence. The software and detailed results for individual genomes are freely available on request.

A qRT-PCR-based method for the measurement ofrrnoperon copy number

To develop a convenient and accurate method for estimating the rrn operon copy number (Y(rrn)) in cells of pure prokaryotic cultures based on quantitative real-time polymerase chain reaction (qRT-PCR). Using Escherichia coli, the Y(rrn) of which is known to be 7, as a reference, the rrn concentrations of target species and E. coli in sample solutions were measured based on their respective threshold cycle numbers (C(t)), whereas the cell concentrations of both species were measured by microscopic counting after staining. The Y(rrn) of the target species was then calculated from the initial cell concentrations and the rrn concentrations of the target species and E. coli. Using this method, the Y(rrn) values of four species, i.e. Xanthomonas campestris, Staphylococcus aureus, Aeromonas hydrophila and Pseudomonas fluorescens, were estimated as 1.80, 4.73, 8.58 and 5.13, respectively, comparable to their respective known values of 2, 5, 10, and 5, resulting in an average deviation of 8%. The whole cell qRT-PCR based methods were convenient, accurate and reproducible in quantification of rrn copy number of prokaryotic cells. qTR-PCR is a fast and reliable DNA quantification approach. Compared with previous qTR-PCR based methods measuring rrn copy number, the present method avoided the prerequisite for the information on genome size and GC content of target bacteria or a gene with known copy number, thus should be more widely applicable.

Codon-Optimized Fluorescent Proteins Designed for Expression in Low-GC Gram-Positive Bacteria

Fluorescent proteins have wide applications in biology. However, not all of these proteins are properly expressed in bacteria, especially if the codon usage and genomic GC content of the host organism are not ideal for high expression. In this study, we analyzed the DNA sequences of multiple fluorescent protein genes with respect to codons and GC content and compared them to a low-GC gram-positive bacterium, Bacillus anthracis. We found high discrepancies for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the photoactivatable green fluorescent protein (PAGFP), but not GFP, with regard to GC content and codon usage. Concomitantly, when the proteins were expressed in B. anthracis, CFP- and YFP-derived fluorescence was undetectable microscopically, a phenomenon caused not by lack of gene transcription or degradation of the proteins but by lack of protein expression. To improve expression in bacteria with low genomic GC contents, we synthesized a codon-optimized gfp and constructed optimized photoactivatable pagfp, cfp, and yfp, which were in contrast to nonoptimized genes highly expressed in B. anthracis and in another low-GC gram-positive bacterium, Staphylococcus aureus. Using optimized GFP as a reporter, we were able to monitor the activity of the protective antigen promoter of B. anthracis and confirm its dependence on bicarbonate and regulators present on virulence plasmid pXO1.

Codon and Aminoacid Usage Patterns in Mycobacteria

The large number of organisms and of genes sequenced at the present time permits now to study molecular evolution in such lower clades as genera, species, and subspecies. Here, we focus our attention on the genus Mycobacterium, in which we examined codon and aminoacid usage in 13 species, and in 12 subspecies for a total of 8,836,513 codons from 26,755 sequences. Within the genus Mycobacterium, frequencies of codon and aminoacid usage correlate between species and between subspecies. In the groups studied, aminoacid molecular weight and codon degeneracy influence correlations between frequencies, while GC content is the main factor influencing the effective number of codons. The coding GC, which is highly correlated with total genomic GC content, seems to be the main factor influencing present synonymous codon usage in the genus. In particular, the GC content at the 3rd base position seems to shape heavily the effective number of codons, giving indication that here mutational bias dominates over translational selection. Evolutionary trees based on codon and aminoacid usage are consistent with traditional phylogenies of species within the genus.

Nucleotide Composition and Amino Acid Usage in AT-Rich Hyperthermophilic Species

Nucleotide composition, codon usage and amino acid content are important molecular signatures that vary in different groups of organisms. AT-rich (or GC poor) hyperthermophiles have relatively been unexplored in these aspects. In this study, we have examined the compositional characteristics of AT rich genomes viz. Methanococcus jannaschii, Sulfolobus solfataricus, Sulfolobus tokodaii and Nanoarcheum equitans by their comparison with four mesophiles having similar genomic GC content. The analysis revealed a significant increase in purine content of ORFs due to increase in guanine content. Moreover, the influence of dinucleotide composition on protein thermostability was found even larger. Accordingly, increased usage of codons that are constituted of dinucleotides RR was observed. Arginine, proline, valine and tyrosine were most abundant amino acids in hyperthermophilic proteomes, and similar bias was seen when dipeptidic composition of proteins was compared. Further amino acid composition analysis of alpha helices indicates an increased usage of E, K, R and decreased usage of N and Q. Summing up, the study suggested that elevated growth temperature im- pose selective constraints at all the three molecular levels- nucleotide composition, codon usage and amino acid content.

Reliability and applications of statistical methods based on oligonucleotide frequencies in bacterial and archaeal genomes

BackgroundThe increasing number of sequenced prokaryotic genomes contains a wealth of genomic data that needs to be effectively analysed. A set of statistical tools exists for such analysis, but their strengths and weaknesses have not been fully explored. The statistical methods we are concerned with here are mainly used to examine similarities between archaeal and bacterial DNA from different genomes. These methods compare observed genomic frequencies of fixed-sized oligonucleotides with expected values, which can be determined by genomic nucleotide content, smaller oligonucleotide frequencies, or be based on specific statistical distributions. Advantages with these statistical methods include measurements of phylogenetic relationship with relatively small pieces of DNA sampled from almost anywhere within genomes, detection of foreign/conserved DNA, and homology searches. Our aim was to explore the reliability and best suited applications for some popular methods, which include relative oligonucleotide frequencies (ROF), di- to hexanucleotide zero'th order Markov methods (ZOM) and 2.order Markov chain Method (MCM). Tests were performed on distant homology searches with large DNA sequences, detection of foreign/conserved DNA, and plasmid-host similarity comparisons. Additionally, the reliability of the methods was tested by comparing both real and random genomic DNA.ResultsOur findings show that the optimal method is context dependent. ROFs were best suited for distant homology searches, whilst the hexanucleotide ZOM and MCM measures were more reliable measures in terms of phylogeny. The dinucleotide ZOM method produced high correlation values when used to compare real genomes to an artificially constructed random genome with similar %GC, and should therefore be used with care. The tetranucleotide ZOM measure was a good measure to detect horizontally transferred regions, and when used to compare the phylogenetic relationships between plasmids and hosts, significant correlation (R2 = 0.4) was found with genomic GC content and intra-chromosomal homogeneity.ConclusionThe statistical methods examined are fast, easy to implement, and powerful for a number of different applications involving genomic sequence comparisons. However, none of the measures examined were superior in all tests, and therefore the choice of the statistical method should depend on the task at hand.