Bayesian modeling of recombination events in bacterial populations

Background  

We consider the discovery of recombinant segments jointly with their origins within multilocus DNA sequences from bacteria representing heterogeneous populations of fairly closely related species. The currently available methods for recombination detection capable of probabilistic characterization of uncertainty have a limited applicability in practice as the number of strains in a data set increases.     

Detection of homologous recombination events in SARS-CoV-2

Biotechnology Letters - The COVID-19 disease with acute respiratory symptoms emerged in 2019. The causal agent of the disease, the SARS-CoV-2 virus, is classified into the Betacoronaviruses family....     

Dynamics of mutation and recombination in a replicating population of complementing, defective viral genomes

In a previous study, we documented that serial passage of a biological clone of foot-and-mouth disease virus (FMDV) at high multiplicity of infection (moi) in cell culture resulted in viral populations dominated by defective genomes that included internal in-frame deletions, affecting the L and capsid-coding regions, and were infectious by complementation. In the present study, analyses of the defective genomes present in individual viral plaques, and of consensus nucleotide sequences determined for the entire genomes of sequential samples, have revealed a continuous dynamics of mutation and recombination. At some points of high genetic instability, multiple minority genomes with different internal deletions co-existed in the population. At later passages, a new defective RNA arose and displaced a related, previously dominant RNA. Nucleotide sequences of the different genomic forms found in sequential isolates have revealed an accumulation of mutations at an average rate of 0.12 substitutions per genome per passage. At the regions around the deletion sites, substantial, minor or no nucleotide sequence identity is found, suggesting relaxed sequence requirements for the occurrence of internal deletions. Competition experiments indicate a selective advantage of late phase defective genomes over their precursor forms. The defective genome-based FMDV retained an expansion of host cell tropism, undergone by the standard virus at a previous stage of the same evolutionary lineage. Thus, despite a complex dynamics of mutation and recombination, and phases of high genetic instability, a biologically relevant phenotypic trait was stably maintained after the evolutionary transition towards a primitive genome segmentation. The results extend the concept of a complex spectrum of mutant genomes to a complex spectrum of defective genomes in some evolutionary transitions of RNA viruses.     

Detection of bacterial pathogens in environmental samples using DNA microarrays

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.     

Spectrum: joint Bayesian inference of population structure and recombination events

MOTIVATION: While genetic properties such as linkage disequilibrium (LD) and population structure are closely related under a common inheritance process, the statistical methodologies developed so far mostly deal with LD analysis and structural inference separately, using specialized models that do not capture their statistical and genetic relationships. Also, most of these approaches ignore the inherent uncertainty in the genetic complexity of the data and rely on inflexible models built on a closed genetic space. These limitations may make it difficult to infer detailed and consistent structural information from rich genomic data such as populational single nucleotide polymorphisms (SNP) profiles. RESULTS: We propose a new model-based approach to address these issues through joint inference of population structure and recombination events under a non-parametric Bayesian framework; we present Spectrum, an efficient implementation based on our new model. We validated Spectrum on simulated data and applied it to two real SNP datasets, including single-population Daly data and the four-population HapMap data. Our method performs well relative to LDhat 2.0 in estimating the recombination rates and hotspots on these datasets. More interestingly, it generates an ancestral spectrum for representing population structures which not only displays sub-structure based on population founders but also reveals details of the genetic diversity of each individual. It offers an alternative view of the population structures to that offered by Structure 2.1, which ignores chromosome-level mutation and recombination with respect to founders.     

Evolution of bacterial genomes

This review examines evolution of bacterial genomes with an emphasis on RNA based life, the transition to functional DNA and small evolving genomes (possibly plasmids) that led to larger, functional bacterial genomes.     

Site-specific recombination system based on actinophage TG1 integrase for gene integration into bacterial genomes

Phage integrases are enzymes that catalyze unidirectional site-specific recombination between the attachment sites of phage and host bacteria, attP and attB, respectively. We recently developed an in vivo intra-molecular site-specific recombination system based on actinophage TG1 serine-type integrase that efficiently acts between attP and attB on a single plasmid DNA in heterologous Escherichia coli cells. Here, we developed an in vivo inter-molecular site-specific recombination system that efficiently acted between the att site on exogenous non-replicative plasmid DNA and the corresponding att site on endogenous plasmid or genomic DNA in E. coli cells, and the recombination efficiencies increased by a factor of ~10^1–3 in cells expressing TG1 integrase over those without. Moreover, integration of attB-containing incoming plasmid DNA into attP-inserted E. coli genome was more efficient than that of the reverse substrate configuration. Together with our previous result that purified TG1 integrase functions efficiently without auxiliary host factors in vitro, these in vivo results indicate that TG1 integrase may be able to introduce attB-containing circular DNAs efficiently into attP-inserted genomes of many bacterial species in a site-specific and unidirectional manner. This system thus may be beneficial to genome engineering for a wide variety of bacterial species.     

Hybrid selection for sequencing pathogen genomes from clinical samples

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla.     

Gene recognition from questionable ORFs in bacterial and archaeal genomes

The ORFs of microbial genomes in annotation files are usually classified into two groups: the first corresponds to known genes; whereas the second includes 'putative', 'probable', 'conserved hypothetical', 'hypothetical', 'unknown' and 'predicted' ORFs etc. Since the annotation is not 100% accurate, it is essential to confirm which ORF of the latter group is coding and which is not. Starting from known genes in the former, this paper describes an improved Z curve method to recognize genes in the latter. Ten-fold cross-validation tests show that the average accuracy of the algorithm is greater than 99% for recognizing the known genes in 57 bacterial and archaeal genomes. The method is then applied to recognize genes of the latter group. The likely non-coding ORFs in each of the 57 bacterial or archaeal genomes studied here are recognized and listed at the website http://tubic.tju.edu.cn/ZCURVE_C_html/noncoding.html. The working mechanism of the algorithm has been discussed in details. A computer program, called ZCURVE_C, was written to calculate a coding score called Z-curve score for ORFs in the above 57 bacterial and archaeal genomes. Coding/non-coding is simply determined by the criterion of Z-curve score > 0/ Z-curve score < 0. A website has been set up to provide the service to calculate the Z-curve score. A user may submit the DNA sequence of an ORF to the server at http://tubic.tju.edu.cn/ZCURVE_C/Default.cgi, and the Z-curve score of the ORF is calculated and returned to the user immediately.     

Automated sequencing of complete mitochondrial genomes from laser-capture microdissected samples

Mitochondrial DNA mutations have been related to both aging and a variety of diseases such as cancer. Due to the relatively small size of the genome (16 kb) and with the use of automated DNA sequencing, the entire genome can be sequenced from clinical specimens in days. We present a reliable approach to complete mitochondrial genome sequencing from laser-capture microdissected human clinical cancer specimens that overcome the inherent limitations of relatively small tissue samples and partial DNA degradation, which are unavoidable when laser-capture microdissection is used to attain pure populations of cells from heterogeneous tissues obtained from surgical procedures. The acquisition of sufficient template combined with a standard set of 18 pairs of PCR primers allows for the efficient amplification of the genome. Subsequent single-stranded amplification is performed using 36 sequencing primers, and samples are run on an ABI PRISM 3100 Genetic Analyzer. The use of this procedure should allow even investigators with little experience sequencing from clinical specimens success in complete mitochondrial genome sequencing.     

Insights into recombination from population genetic variation

Patterns of genetic variation in natural populations are shaped by, and hence carry valuable information about, the underlying recombination process. In the past five years, the increasing availability of large-scale population genetic data on dense sets of markers, coupled with advances in statistical methods for extracting information from these data, have led to several important advances in our understanding of the recombination process in humans. These advances include the identification of large numbers of 'hotspots', where recombination appears to take place considerably more frequently than in the surrounding sequence, and the identification of DNA sequence motifs that are associated with the locations of these hotspots.     

Recognizing the pseudogenes in bacterial genomes

Pseudogenes are now known to be a regular feature of bacterial genomes and are found in particularly high numbers within the genomes of recently emerged bacterial pathogens. As most pseudogenes are recognized by sequence alignments, we use newly available genomic sequences to identify the pseudogenes in 11 genomes from 4 bacterial genera, each of which contains at least 1 human pathogen. The numbers of pseudogenes range from 27 in Staphylococcus aureus MW2 to 337 in Yersinia pestis CO92 (e.g. 1–8% of the annotated genes in the genome). Most pseudogenes are formed by small frameshifting indels, but because stop codons are A + T-rich, the two low-G + C Gram-positive taxa (Streptococcus and Staphylococcus) have relatively high fractions of pseudogenes generated by nonsense mutations when compared with more G + C-rich genomes. Over half of the pseudogenes are produced from genes whose original functions were annotated as ‘hypothetical’ or ‘unknown’; however, several broadly distributed genes involved in nucleotide processing, repair or replication have become pseudogenes in one of the sequenced Vibrio vulnificus genomes. Although many of our comparisons involved closely related strains with broadly overlapping gene inventories, each genome contains a largely unique set of pseudogenes, suggesting that pseudogenes are formed and eliminated relatively rapidly from most bacterial genomes.     

Selection for unequal densities of sigma70 promoter-like signals in different regions of large bacterial genomes

The evolutionary processes operating in the DNA regions that participate in the regulation of gene expression are poorly understood. In Escherichia coli, we have established a sequence pattern that distinguishes regulatory from nonregulatory regions. The density of promoter-like sequences, that could be recognizable by RNA polymerase and may function as potential promoters, is high within regulatory regions, in contrast to coding regions and regions located between convergently transcribed genes. Moreover, functional promoter sites identified experimentally are often found in the subregions of highest density of promoter-like signals, even when individual sites with higher binding affinity for RNA polymerase exist elsewhere within the regulatory region. In order to see the generality of this pattern, we have analyzed 43 additional genomes belonging to most established bacterial phyla. Differential densities between regulatory and nonregulatory regions are detectable in most of the analyzed genomes, with the exception of those that have evolved toward extreme genome reduction. Thus, presence of this pattern follows that of genes and other genomic features that require weak selection to be effective in order to persist. On this basis, we suggest that the loss of differential densities in the reduced genomes of host-restricted pathogens and symbionts is an outcome of the process of genome degradation resulting from the decreased efficiency of purifying selection in highly structured small populations. This implies that the differential distribution of promoter-like signals between regulatory and nonregulatory regions detected in large bacterial genomes confers a significant, although small, fitness advantage. This study paves the way for further identification of the specific types of selective constraints that affect the organization of regulatory regions and the overall distribution of promoter-like signals through more detailed comparative analyses among closely related bacterial genomes.     

Order and disorder in bacterial genomes

The availability of sequenced bacterial genomes allows a deeper understanding of their organizational features that are related with fundamental cellular processes such as coordinated gene expression, chromosome replication and cell division. Nevertheless, recent genome comparisons and experimental work highlighted the fluidity of bacterial chromosomes, including genome rearrangements that imperil the selective features of chromosome order. As a result, the clash between elements generating rearrangements and chromosome organization is a classic case of evolutionary conflict.     

The evolution of domain-content in bacterial genomes

Background  

Across all sequenced bacterial genomes, the number of domains n _c in different functional categories c scales as a power-law in the total number of domains n, i.e. , with exponents α _c that vary across functional categories. Here we investigate the implications of these scaling laws for the evolution of domain-content in bacterial genomes and derive the simplest evolutionary model consistent with these scaling laws.     

The generation of variation in bacterial genomes

In the context of a general overview of molecular mechanisms of microbial evolution, several genetic systems known to either promote or restrain the generation of genetic variations are discussed. Particular attention is given to functions involved in DNA rearrangements and DNA acquisition. Sporadic actions by a variety of such systems influencing genetic stability in either way result in a level of genetic plasticity which is tolerable to the overall wealth of microbial populations but which allows for evolutionary change needed for a steady adaptation to variable selective forces. Although these evolutionarily relevant biological functions are encoded by the genome of each individual, their actions are exerted to some degree randomly in rare individuals and are therefore seemingly nondeterministic and become manifest at the population level.     

Absence of interparental recombination in multiplicity reconstitution from incomplete bacteriophage T4 genomes.

Interparental recombination between injected T4 DNA molecules is indetectable for incomplete petite phages (carrying a terminally deficient genome and therefore unable to circularize) as well as for genetically complete phages. The nonvialbe petite phages can individually replicate their DNA repeatedly, and they aso undergo multiplicity reconstitution, producing complete phages, provided that a host bacterium is infected by several petite particles that carry genetically complementary segments of DNA. The formation of complete phages in multiplicity reconstitution must be due to recombination among incomplete progeny fragments, i.e., partial replicas of the T4 genomes. It evidently does not result from interparental recombination. To test for interparental recombination, light bacteria (containing no bromouracil) were simultaneously infected in light medium with light radioactive phage in minority (usually less than one per cell) and heavy (bromouracil-labeled) phage in majority (usually about nine per cell). Any interparental recombination should, under these circumstances of infection, head to movement of the radioactive label of the minority light phage DNA to a position of higher density. That possibility was not observed.     

Identifying repeat domains in large genomes

We present a graph-based method for the analysis of repeat families in a repeat library. We build a repeat domain graph that decomposes a repeat library into repeat domains, short subsequences shared by multiple repeat families, and reveals the mosaic structure of repeat families. Our method recovers documented mosaic repeat structures and suggests additional putative ones. Our method is useful for elucidating the evolutionary history of repeats and annotating de novo generated repeat libraries.