Biomedical subjects

Genomics

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PubMed2004

Theoretical and practical advances in genome halving.

MOTIVATION: Duplication of an organism's entire genome is a rare but spectacular event, enabling the rapid emergence of multiple new gene functions. Over time, the parallel linkage of duplicated genes across chromosomes may be disrupted by reciprocal translocations, while the intra-chromosomal order of genes may be shuffled by inversions and transpositions. Some duplicate genes may evolve unrecognizably or be deleted. As a consequence, the only detectable signature of an ancient duplication event in a modern genome may be the presence of various chromosomal segments containing parallel paralogous genes, with each segment appearing exactly twice in the genome. The problem of reconstructing the linkage structure of an ancestral genome before duplication is known as genome halving with unordered chromosomes. RESULTS: In this paper, we derive a new upper bound on the genome halving distance that is tighter than the best known, and a new lower bound that is almost always tighter than the best known. We also define the notion of genome halving diameter, and obtain both upper and lower bounds for it. Our tighter bounds on genome halving distance yield a new algorithm for reconstructing an ancestral duplicated genome. We create a software package GenomeHalving based on this new algorithm and test it on the yeast genome, identifying a sequence of translocations for halving the yeast genome that is shorter than previously conjectured possible.

Algorithms↗

PubMed2006

Can a simple algebraic analysis predict markers-genome heterozygosity correlations?

A current algebraic analysis on genome-wide heterozygosity estimates suggests that correlations between molecular markers and genome-wide heterozygosity, rho, depend on the ratio between the number of markers used, r, and the number of genome loci, n; that is: rho approximately square root r/n. Hence, it is unfeasible to obtain reliable estimates of genome-wide heterozygosity in species of large genome using a few markers. We cast some doubts about this analysis as it assumed that the probability that an individual was heterozygous at a locus is equal to the average heterozygosity of this locus in the population. However, we believe that individual heterozygosity at a given locus depends on individual pedigree. Because the pedigree is common for all loci of an individual, their probabilities of heterozygosity are not independent within the genome. We first performed simulations generating random genomes for 100 individuals. Among these individuals, markers and genome-wide heterozygosities correlated as expected from the above equation. However, when we simulated random mating among these individuals and in successive generations including their descendents, as occur in real populations, the correlations between markers and genome-wide heterozygosity were much higher than those predicted from algebraic analyses, and estimates of genome-wide heterozygosity improved slightly with the increment of the number of loci in the genome.

Female↗

PubMed2006

Capturing genomic signatures of DNA sequence variation using a standard anonymous microarray platform.

Comparative genomics, using the model organism approach, has provided powerful insights into the structure and evolution of whole genomes. Unfortunately, only a small fraction of Earth's biodiversity will have its genome sequenced in the foreseeable future. Most wild organisms have radically different life histories and evolutionary genomics than current model systems. A novel technique is needed to expand comparative genomics to a wider range of organisms. Here, we describe a novel approach using an anonymous DNA microarray platform that gathers genomic samples of sequence variation from any organism. Oligonucleotide probe sequences placed on a custom 44 K array were 25 bp long and designed using a simple set of criteria to maximize their complexity and dispersion in sequence probability space. Using whole genomic samples from three known genomes (mouse, rat and human) and one unknown (Gonystylus bancanus), we demonstrate and validate its power, reliability, transitivity and sensitivity. Using two separate statistical analyses, a large numbers of genomic 'indicator' probes were discovered. The construction of a genomic signature database based upon this technique would allow virtual comparisons and simple queries could generate optimal subsets of markers to be used in large-scale assays, using simple downstream techniques. Biologists from a wide range of fields, studying almost any organism, could efficiently perform genomic comparisons, at potentially any phylogenetic level after performing a small number of standardized DNA microarray hybridizations. Possibilities for refining and expanding the approach are discussed.

Animals↗

PubMed2006

Estimation of prokaryote genomic DNA G+C content by sequencing universally conserved genes.

Determination of the DNA G+C content of prokaryotic genomes using traditional methods is time-consuming and results may vary from laboratory to laboratory, depending on the technique used. We explored the possibility of extrapolating the genomic DNA G+C content of prokaryotes from gene sequences. For this, 127 universally conserved genes were studied from 50 prokaryotic genomes in the Clusters of Orthologous Groups database. Of these, 57 genes were present as a single copy in the genomes of 157 different prokaryote species available in GenBank. There was a strong correlation [coefficient of determination (r2) >95 %] between the DNA G+C contents of 20 genes and their corresponding genomes. For each of the 157 prokaryotic genomes studied, the DNA G+C content of the 20 genes was used to determine a 'calculated' genome DNA G+C content (CGC) and this value was compared with the 'real' genome DNA G+C content (RGC). In order to select the most suitable gene for the determination of CGC values, we compared the r2 and median mol% difference between CGC and RGC as well as the sensitivity of each gene to provide CGC values for prokaryotic genomes that differ by less than 5 mol% from their RGC. The highly conserved ftsY gene (median size 1144 nucleotides), a vertically inherited member of the GTPase superfamily, showed the highest r2 value of 0.98, the smallest median mol% difference between CGC and RGC of 1.06 and a sensitivity of 100 %. Using ftsY DNA G+C content values, the CGC values of 100 genomes not included in the calculation of r2 differed by less than 5 mol% from their RGC values. These data suggest that the genomic DNA G+C content of prokaryotes may be estimated easily and reliably from the ftsY gene sequence.

Bacteria↗

PubMed2004

Mauve: multiple alignment of conserved genomic sequence with rearrangements.

As genomes evolve, they undergo large-scale evolutionary processes that present a challenge to sequence comparison not posed by short sequences. Recombination causes frequent genome rearrangements, horizontal transfer introduces new sequences into bacterial chromosomes, and deletions remove segments of the genome. Consequently, each genome is a mosaic of unique lineage-specific segments, regions shared with a subset of other genomes and segments conserved among all the genomes under consideration. Furthermore, the linear order of these segments may be shuffled among genomes. We present methods for identification and alignment of conserved genomic DNA in the presence of rearrangements and horizontal transfer. Our methods have been implemented in a software package called Mauve. Mauve has been applied to align nine enterobacterial genomes and to determine global rearrangement structure in three mammalian genomes. We have evaluated the quality of Mauve alignments and drawn comparison to other methods through extensive simulations of genome evolution.

Chromosomes, Bacterial↗

PubMed2004

Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL/6J, as defined by BAC-end sequence-SNP analysis.

MSM/Ms is an inbred strain derived from the Japanese wild mouse, Mus musculus molossinus. It is believed that subspecies molossinus has contributed substantially to the genome constitution of common laboratory strains of mice, although the majority of their genome is derived from the west European M. m. domesticus. Information on the molossinus genome is thus essential not only for genetic studies involving molossinus but also for characterization of common laboratory strains. Here, we report the construction of an arrayed bacterial artificial chromosome (BAC) library from male MSM/Ms genomic DNA, covering approximately 1x genome equivalent. Both ends of 176,256 BAC clone inserts were sequenced, and 62,988 BAC-end sequence (BES) pairs were mapped onto the C57BL/6J genome (NCBI mouse Build 30), covering 2,228,164 kbp or 89% of the total genome. Taking advantage of the BES map data, we established a computer-based clone screening system. Comparison of the MSM/Ms and C57BL/6J sequences revealed 489,200 candidate single nucleotide polymorphisms (SNPs) in 51,137,941 bp sequenced. The overall nucleotide substitution rate was as high as 0.0096. The distribution of SNPs along the C57BL/6J genome was not uniform: The majority of the genome showed a high SNP rate, and only 5.2% of the genome showed an extremely low SNP rate (percentage identity = 0.9997); these sequences are likely derived from the molossinus genome.

Animals↗

PubMed2002

Genome organization and reorganization in evolution: formatting for computation and function.

This volume deals with the role of epigenetics in life and evolution. The most dynamic forms of functional genome formatting involve DNA interacting with cellular complexes that do not alter sequence information. Such important epigenetic phenomena are the main subjects of other articles in this volume. This article focuses on the long-lived form of genome formatting that lies within the DNA sequence itself. I argue for a computational view of genome function as the long-term information storage organelle of each cell. Structural formatting consists of organizing various signals and coding sequences into computationally ready systems facilitating genome expression and genome transmission. The basic features of genome organization can be understood by examining the E. coli lac operon as a paradigmatic genomic system. Multiple systems are connected through distributed signals and repetitive DNA to form higher-order genome system architectures. Molecular discoveries about mechanisms of DNA restructuring show that cells possess the natural genetic engineering functions necessary for evolutionary change by rearranging genomic components and reorganizing system architectures. The concepts of cellular computation and decision-making, genome system architecture, and natural genetic engineering combine to provide a new way of framing evolutionary theories and understanding genome sequence information.

Animals↗

PubMed2002

The evolving genome of Salmonella enterica serovar Pullorum.

Salmonella enterica serovar Pullorum is a fowl-adapted bacterial pathogen that causes dysentery (pullorum disease). Host adaptation and special pathogenesis make S. enterica serovar Pullorum an exceptionally good system for studies of bacterial evolution and speciation, especially regarding pathogen-host interactions and the acquisition of pathogenicity. We constructed a genome map of S. enterica serovar Pullorum RKS5078, using I-CeuI, XbaI, AvrII, and SpeI and Tn10 insertions. Pulsed-field gel electrophoresis was employed to separate the large DNA fragments generated by the endonucleases. The genome is 4,930 kb, which is similar to most salmonellas. However, the genome of S. enterica serovar Pullorum RKS5078 is organized very differently from the majority of salmonellas, with three major inversions and one translocation. This extraordinary genome structure was seen in most S. enterica serovar Pullorum strains examined, with different structures in a minority of S. enterica serovar Pullorum strains. We describe the coexistence of different genome structures among the same bacteria as genomic plasticity. Through comparisons with S. enterica serovar Typhimurium, we resolved seven putative insertions and eight deletions ranging in size from 12 to 157 kb. The genomic plasticity seen among S. enterica serovar Pullorum strains supported our hypothesis about its association with bacterial evolution: a large genomic insertion (157 kb in this case) disrupted the genomic balance, and rebalancing by independent recombination events in individual lineages resulted in diverse genome structures. As far as the structural plasticity exists, the S. enterica serovar Pullorum genome will continue evolving to reach a further streamlined and balanced structure.

Chromosome Mapping↗

PubMed1996

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome.

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.

Animals↗

PubMed1999

In vivo addition of poly(A) tail and AU-rich sequences to the 3' terminus of the Sindbis virus RNA genome: a novel 3'-end repair pathway.

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5' end and polyadenylated at its 3' end. The 3' nontranslated region (3'NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3'CSE) and a poly(A) tail. This 3'CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693-2704, 1997). Here, we demonstrate that the 3'-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations in the 3'NTR and tested their ability to generate infectious virus and undergo 3' polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3'CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3'NTR. These results indicate the ability of alphavirus RNAs to undergo 3' repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3' end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.

3' Untranslated Regions↗

PubMed2000

Identification of RAPD markers linked to A and B genome sequences in Musa L.

Plantains and bananas (Musa spp. sect. eumusa) originated from intra- and interspecific hybridization between two wild diploid species, M. acuminata Colla. and M. balbisiana Colla., which contributed the A and B genomes, respectively. Polyploidy and hybridization have given rise to a number of diploid, triploid, and tetraploid clones with different permutations of the A and B genomes. Thus, dessert and highland bananas are classified mainly as AAA, plantains are AAB, and cooking bananas are ABB. Classification of Musa into genomic groups has been based on morphological characteristics. This study aimed to identify RAPD (random amplified polymorphic DNA) markers for the A and B genomes. Eighty 10-mer Operon primers were used to amplify DNA from M. acuminata subsp. burmannicoides clone 'Calcutta 4' (AA genomes) and M. balbisiana clone 'Honduras' (BB genomes). Three primers (A17, A18, and D10) that produced unique genome-specific fragments in the two species were identified. These primers were tested in a sample of 40 genotypes representing various genome combinations. The RAPD markers were able to elucidate the genome composition of all the genotypes. The results showed that RAPD analysis can provide a quick and reliable system for genome identification in Musa that could facilitate genome characterization and manipulations in breeding lines.

Animals↗

PubMed2006

Compositional bias and size of genomes of human DNA viruses.

Genomes of 144 human DNA viruses were analyzed in the aspect of their compositional asymmetry. DNA viruses were divided into two groups according to their genome sizes. The analysis revealed that the level of guanine and cytosine (GC content) in the coding sequences of small genome DNA viruses was significantly lower than that of large genome DNA viruses. Because small genome viruses replicate their genomes using cellular enzymes, while large genome viruses use their own enzymes for genome replication, the two groups of viruses may be under different mutational bias and/or selection pressure. In these viruses, GC content at the third codon position correlated with GC content at the first and second codon position. However, the relationship in small genome DNA viruses was weaker than that in large genome DNA viruses, suggesting that their genome composition may be more strongly influenced by codon usage preference or restriction on amino acid composition.

Base Composition↗

PubMed1991

Genetic relationship between thirteen genome types of adenovirus 11, 34, and 35 with different tropisms.

Eleven genome types of adenovirus serotype 11 (Ad11) were identified among 20 strains isolated from healthy pregnant women and patients with urinary tract infections, respiratory tract infections, or pharyngoconjunctival fever by use of 13 restriction endonucleases: BamHI, BclI, BglI, BglII, BstEII, EcoRI, HindIII, HpaI, PstI, SalI, SmaI, XbaI, and XhoI. All genome types could be grouped into three genomic clusters according to their genetic homology expressed as pairwise comigrating restriction fragments. The genome types within a genomic cluster were very closely related. They shared on an average pairwise comigrating restriction fragments of 91.6-97.7%. The Ad11 strains of genomic clusters 1 and 3 were isolated from urine, whereas all the Ad11 strains isolated from the respiratory tract were identified as members of the genomic cluster 2. One genome type of Ad34 and one genome type of Ad35 were identified from a hemorrhagic cystitis patient and an organ transplant recipient, respectively. Both were closely related to Ad11. The genome type of Ad35 could be located in the Ad11 genomic cluster 1.

Adenoviruses, Human↗

PubMed2004

GenomeViz: visualizing microbial genomes.

BACKGROUND: An increasing number of microbial genomes are being sequenced and deposited in public databases. In addition, several closely related strains are also being sequenced in order to understand the genetic basis of diversity and mechanisms that lead to the acquisition of new genetic traits. These exercises have necessitated the requirement for visualizing microbial genomes and performing genome comparisons on a finer scale. We have developed GenomeViz to enable rapid visualization and subsequent comparisons of several microbial genomes in an interactive environment. RESULTS: Here we describe a program that allows visualization of both qualitative and quantitative information from complete and partially sequenced microbial genomes. Using GenomeViz, data deriving from studies on genomic islands, gene/protein classifications, GC content, GC skew, whole genome alignments, microarrays and proteomics may be plotted. Several genomes can be visualized interactively at the same time from a comparative genomic perspective and publication quality circular genome plots can be created. CONCLUSIONS: GenomeViz should allow researchers to perform visualization and comparative analysis of up to eight different microbial genomes simultaneously.

Bacterial Proteins↗

PubMed2005

Systematic determination of the mosaic structure of bacterial genomes: species backbone versus strain-specific loops.

BACKGROUND: Public databases now contain multitude of complete bacterial genomes, including several genomes of the same species. The available data offers new opportunities to address questions about bacterial genome evolution, a task that requires reliable fine comparison data of closely related genomes. Recent analyses have shown, using pairwise whole genome alignments, that it is possible to segment bacterial genomes into a common conserved backbone and strain-specific sequences called loops. RESULTS: Here, we generalize this approach and propose a strategy that allows systematic and non-biased genome segmentation based on multiple genome alignments. Segmentation analyses, as applied to 13 different bacterial species, confirmed the feasibility of our approach to discern the 'mosaic' organization of bacterial genomes. Segmentation results are available through a Web interface permitting functional analysis, extraction and visualization of the backbone/loops structure of documented genomes. To illustrate the potential of this approach, we performed a precise analysis of the mosaic organization of three E. coli strains and functional characterization of the loops. CONCLUSION: The segmentation results including the backbone/loops structure of 13 bacterial species genomes are new and available for use by the scientific community at the URL: http://genome.jouy.inra.fr/mosaic.

Agrobacterium tumefaciens↗

PubMed2006

Genome-wide DNA polymorphism analyses using VariScan.

BACKGROUND: DNA sequence polymorphisms analysis can provide valuable information on the evolutionary forces shaping nucleotide variation, and provides an insight into the functional significance of genomic regions. The recent ongoing genome projects will radically improve our capabilities to detect specific genomic regions shaped by natural selection. Current available methods and software, however, are unsatisfactory for such genome-wide analysis. RESULTS: We have developed methods for the analysis of DNA sequence polymorphisms at the genome-wide scale. These methods, which have been tested on a coalescent-simulated and actual data files from mouse and human, have been implemented in the VariScan software package version 2.0. Additionally, we have also incorporated a graphical-user interface. The main features of this software are: i) exhaustive population-genetic analyses including those based on the coalescent theory; ii) analysis adapted to the shallow data generated by the high-throughput genome projects; iii) use of genome annotations to conduct a comprehensive analyses separately for different functional regions; iv) identification of relevant genomic regions by the sliding-window and wavelet-multiresolution approaches; v) visualization of the results integrated with current genome annotations in commonly available genome browsers. CONCLUSION: VariScan is a powerful and flexible suite of software for the analysis of DNA polymorphisms. The current version implements new algorithms, methods, and capabilities, providing an important tool for an exhaustive exploratory analysis of genome-wide DNA polymorphism data.

Algorithms↗

PubMed2006

M-GCAT: interactively and efficiently constructing large-scale multiple genome comparison frameworks in closely related species.

BACKGROUND: Due to recent advances in whole genome shotgun sequencing and assembly technologies, the financial cost of decoding an organism's DNA has been drastically reduced, resulting in a recent explosion of genomic sequencing projects. This increase in related genomic data will allow for in depth studies of evolution in closely related species through multiple whole genome comparisons. RESULTS: To facilitate such comparisons, we present an interactive multiple genome comparison and alignment tool, M-GCAT, that can efficiently construct multiple genome comparison frameworks in closely related species. M-GCAT is able to compare and identify highly conserved regions in up to 20 closely related bacterial species in minutes on a standard computer, and as many as 90 (containing 75 cloned genomes from a set of 15 published enterobacterial genomes) in an hour. M-GCAT also incorporates a novel comparative genomics data visualization interface allowing the user to globally and locally examine and inspect the conserved regions and gene annotations. CONCLUSION: M-GCAT is an interactive comparative genomics tool well suited for quickly generating multiple genome comparisons frameworks and alignments among closely related species. M-GCAT is freely available for download for academic and non-commercial use at: http://alggen.lsi.upc.es/recerca/align/mgcat/intro-mgcat.html.

Algorithms↗

PubMed2004

Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR.

BACKGROUND: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. RESULTS: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively. CONCLUSION: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks.

Bacterial Typing Techniques↗