Use of a multilocus variable-number tandem repeat analysis method for molecular subtyping and phylogenetic analysis of Neisseria meningitidis isolates

Background  

The multilocus variable-number tandem repeat (VNTR) analysis (MLVA) technique has been developed for fine typing of many bacterial species. The genomic sequences of Neisseria meningitidis strains Z2491, MC58 and FAM18 have been available for searching potential VNTR loci by computer software. In this study, we developed and evaluated a MLVA method for molecular subtyping and phylogenetic analysis of N. meningitidis strains.     

Molecular characterization of Korean Bacillus anthracis isolates by amplified fragment length polymorphism analysis and multilocus variable-number tandem repeat analysis

We analyzed the genetic relationships and molecular characteristics of 34 Bacillus anthracis isolates from soil and clinical samples in various regions of Korea and 17 related Bacillus species, using the amplified fragment length polymorphism (AFLP) and multilocus variable-number tandem repeat (MLVA) approaches. Triplicate AFLP profiles of these strains showed high reproducibility and identified 376 polymorphisms. AFLP phylogenetic analysis of B. anthracis isolates showed a high level of similarity, 0.93, and this monomorphic fragment profile proved to be useful to differentiate B. anthracis strains from other Bacillus species. The B. cereus group was separated from other Bacillus species at a level of similarity of 0.68. Among them, some B. cereus strains showed genetic interspersion with B. thuringiensis strains. The evolutionary pattern of nucleotide differences among B. anthracis strains with the eight MLVA markers showed nine MLVA types. Three MLVA types, M1 to M3, were pathogenic B. anthracis isolates and were assigned as new genotypes belonging to the A4 and B3 clusters, compared with 89 genotypes deduced from previous data. This indicates that differences in cluster prevalence and distribution may be influenced more by MLVA markers on two plasmids loci and human activity. Consequently, we suggest that the novel MLVA type may represent significant evidence for historic adaptation to environmental conditions of the Asian continent, particularly Korea. Therefore, MLVA techniques may be available for molecular monitoring on anthrax-release-related bioterrorism and further study is required for the continuous epidemiological study of variable anthrax collections.     

Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay

The Escherichia coli (E. coli) reference collection, ECOR, consists of 72 strains that are representative of the genotypic diversity, as indexed by multilocus enzyme electrophoresis (MLEE), in the species as a whole. MLEE revealed 4 main phylogenetic groups designated A, B1, B2 and D. We present a study of the relationship between the ECOR strains as determined by polymorphisms in seven variable-number of tandem repeats (VNTR) loci. Seven tandem repeats that were present in more than one of the fully sequenced E. coli strains were selected, and primers were constructed in order to amplify the targets in all species where the loci were present. The combined result for all VNTR loci was adapted as a multiple-locus variable-number tandem repeats analysis (MLVA) and showed that the ECOR collection was divided into 63 distinct genotypes. The ECOR phylogenetic groups defined by MLEE were not well conserved by MLVA. A set of 61 pathogenic isolates of both E. coli and Shigella spp. was then tested with the same set of VNTR loci, and revealed 54 distinct genotypes. In addition, the MLVA method was used to genotype isolates from patients and suspected sources in a recent outbreak of E. coli O103 in Norway. The pathogenic E. coli isolates contained the diarrhea causing categories EIEC, EAEC, STEC, ETEC and EPEC. Shigella isolates were of species S. flexneri, S. boydii, S. sonnei and S. dysenteriae. The MLVA method rapidly genotyped all isolates in the study at a Simpson's index of diversity of D=0.98.     

Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi

Thus far, genotyping of Enterocytozoon bieneusi has been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes of E. bieneusi have been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database of E. bieneusi isolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenic E. bieneusi genotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonotic E. bieneusi genotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most E. bieneusi genotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., the public health significance of E. bieneusi in animals, and the sources of human E. bieneusi infections.     

Genotools SNP manager: a new software for automated high-throughput MALDI-TOF mass spectrometry SNP genotyping

Analysis of single nucleotide polymorphisms (SNPs) is a rapidly growing field of research that provides insights into the most common type of differences between individual genomes. The resulting information has a strong impact in the fields of pharmacogenomics, drug development, forensic medicine, and diagnostics of specific disease markers. The technique of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) has been shown to be a highly suitable tool for the analysis of DNA. It supplies a very versatile method for addressing a high-throughput SNP genotyping approach. Here, we present the Bruker genotools SNP MANAGER, a new software tool suitable for highly automated MALDI-TOF MS SNP genotyping. The genotools SNP MANAGER administers the sample preparation data, calculates masses of allele-specific primer extension products, performs genotyping analysis, and displays the results. In the current study, we have used the genotools SNP MANAGER to perform an automated duplex SNP analysis of two biallelic markers from the promoter of the gene encoding the inflammatory mediator interleukin-6.     

New generation sequencers as a tool for genotyping of highly polymorphic multilocus MHC system

Accurate genotyping of complex systems, such as the major histocompatibility complex (MHC) often requires simultaneous analysis of multiple co-amplifying loci. Here we explore the utility of the massively parallel 454 sequencing method as a universal tool for genotyping complex MHC systems in nonmodel vertebrates. The power of this approach stems from the use of tagged polymerase chain reaction (PCR) primers to identify individual amplicons which can be simultaneously sequenced to the arbitrarily chosen coverage. However, the error-prone sequencing technology poses considerable challenges as it may be difficult to discriminate between sequencing errors and true rare alleles; due to complex nature of artefacts and errors, efficient quality control is required. Nevertheless, our study demonstrates the parallel 454 sequencing can be an efficient genotyping platform for MHC and provides an alternative to classical genotyping methods. We introduced procedures to identify the threshold that can be used to reduce number of genotyping errors by eliminating most of artefactual alleles (AA) representing PCR or sequencing errors. Our procedures are based on two expectations: first, that AA should be relatively rare, both overall and on per-individual basis, and second, that most AA result from errors introduced to sequences of true alleles. In our data set, alleles with an average per-individual frequency below 3% most likely represented artefacts. This threshold will vary in other applications according to the complexity of the genotyped system. We strongly suggest direct assessment of genotyping error in every experiment by running a fraction of duplicates: individuals amplified in independent PCRs.     

Design of a multiplex PCR for genotyping 16 short tandem repeats in degraded DNA samples

The molecular genotyping of individuals and reconstruction of kinship through short and high polymorphic DNA markers, so-called short tandem repeats (STR), has become an important and efficient method in anthropology and forensic science. The here introduced experimental design describes a multiplex PCR capable of simultaneously amplifying 16 STRs and the sex determinant locus amelogenin in a short fragment lengths range from 84 bp to 275 bp. Thus, the design depends predominantly on the routines for DNA typing of historical samples with highly degraded ancient DNA. It is shown, that the newly designed multiplex PCR is suitable for successful typing of both forensic and historical material.     

AccuTyping: new algorithms for automated analysis of data from high-throughput genotyping with oligonucleotide microarrays

Microarray-based analysis of single nucleotide polymorphisms (SNPs) has many applications in large-scale genetic studies. To minimize the influence of experimental variation, microarray data usually need to be processed in different aspects including background subtraction, normalization and low-signal filtering before genotype determination. Although many algorithms are sophisticated for these purposes, biases are still present. In the present paper, new algorithms for SNP microarray data analysis and the software, AccuTyping, developed based on these algorithms are described. The algorithms take advantage of a large number of SNPs included in each assay, and the fact that the top and bottom 20% of SNPs can be safely treated as homozygous after sorting based on their ratios between the signal intensities. These SNPs are then used as controls for color channel normalization and background subtraction. Genotype calls are made based on the logarithms of signal intensity ratios using two cutoff values, which were determined after training the program with a dataset of approximately 160,000 genotypes and validated by non-microarray methods. AccuTyping was used to determine >300,000 genotypes of DNA and sperm samples. The accuracy was shown to be >99%. AccuTyping can be downloaded from http://www2.umdnj.edu/lilabweb/publications/AccuTyping.html.     

MACGT: multi-dimensional automated clustering genotyping tool for analysis of microarray-based mini-sequencing data

SUMMARY: Multi-dimensional Automated Clustering Genotyping Tool (MACGT) is a Java application that clusters complex multi-dimensional vector data derived from single nucleotide polymorphism (SNP) genotyping experiments using mini-sequencing based microarray chemistries such as arrayed primer extension (APEX). Spot intensity output files from microarray experiments across multiple samples are imported into MACGT. The datasets can include four channels of intensity data for each spot, replica spots for each SNP probe and multiple probe types (APEX and allele-specific APEX probes) on both DNA strands for each SNP. MACGT automatically clusters these multi-dimensionality datasets for each SNP across multiple samples. Incorporation of additional array datasets from known samples that have previously validated SNP genotype calls allows unknown samples to be automatically assigned a genotype based on the clustering, along with numerical measures of confidence for each genotype call. Calling accuracy by MACGT exceeds 98% when applied to genotyping data from APEX microarrays, and can be increased to >99.5% by applying thresholds to the confidence measures.     

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.     

Rapid and efficient resolution of parentage by amplification of short tandem repeats.

Short tandem repeat (STR) loci are highly informative polymorphic loci that are gaining popularity for identity testing. We have conducted parentage testing by using nine STR loci on 50 paternity trios that had been previously tested using VNTR loci. These nine unlinked STR loci are amplified in three multiplex reactions and, when examined for genetic informativeness, provide a combined average power of exclusion of 99.73% (Caucasian data). The informative value of the selected loci is based on extensive STR typing of four racial/ethnic populations. In 37 of the 50 cases, paternity could not be excluded by any of the loci. In the remaining 13 cases, paternity was excluded by at least two of the STR markers. The probability of paternity calculated for the alleged father of each matching trio was > 99% in 36 of the 37 inclusion cases. All data agreed with the results reported using VNTR loci and conventional Southern technology. Our studies validate the use of DNA typing with STR loci for parentage testing, thus providing an accurate, highly sensitive, and rapid assay.     

A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

Background  

Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies.     

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.     

Multiple Locus Variable number of tandem repeat Analysis: A molecular genotyping tool for Paenibacillus larvae

American Foulbrood, caused by Paenibacillus larvae, is the most severe bacterial disease of honey bees (Apis mellifera). To perform genotyping of P. larvae in an epidemiological context, there is a need of a fast and cheap method with a high resolution. Here, we propose Multiple Locus Variable number of tandem repeat Analysis (MLVA). MLVA has been used for typing a collection of 209 P. larvae strains from which 23 different MLVA types could be identified. Moreover, the developed methodology not only permits the identification of the four Enterobacterial Repetitive Intergenic Consensus (ERIC) genotypes, but allows also a discriminatory subdivision of the most dominant ERIC type I and ERIC type II genotypes. A biogeographical study has been conducted showing a significant correlation between MLVA genotype and the geographical region where it was isolated.     

CHOMPER: a bioinformatic tool for rapid validation of tandem mass spectrometry search results associated with high-throughput proteomic strategies

Current efforts aimed at developing high-throughput proteomics focus on increasing the speed of protein identification. Although improvements in sample separation, enrichment, automated handling, mass spectrometric analysis, as well as data reduction and database interrogation strategies have done much to increase the quality, quantity and efficiency of data collection, significant bottlenecks still exist. Various separation techniques have been coupled with tandem mass spectrometric (MS/MS) approaches to allow a quicker analysis of complex mixtures of proteins, especially where a high number of unambiguous protein identifications are the exception, rather than the rule. MS/MS is required to provide structural / amino acid sequence information on a peptide and thus allow protein identity to be inferred from individual peptides. Currently these spectra need to be manually validated because: (a) the potential of false positive matches i.e., protein not in database, and (b) observed fragmentation trends may not be incorporated into current MS/MS search algorithms. This validation represents a significant bottleneck associated with high-throughput proteomic strategies. We have developed CHOMPER, a software program which reduces the time required to both visualize and confirm MS/MS search results and generate post-analysis reports and protein summary tables. CHOMPER extracts the identification information from SEQUEST MS/MS search result files, reproduces both the peptide and protein identification summaries, provides a more interactive visualization of the MS/MS spectra and facilitates the direct submission of manually validated identifications to a database.     

Rapid combined genotyping assay for four achondroplasia and hypochondroplasia mutations by real-time PCR with multiple detection probes

Achondroplasia (ACH) and hypochondroplasia (HYCH) are the most prevalent genetic short-stature syndromes. Whereas the diagnosis of ACH can be established on clinical and radiologic grounds alone in the majority of cases, HYCH is more difficult to confirm. Molecular genetic analysis of both skeletal dysplasias can be especially helpful for the purpose of prenatal diagnosis, in early childhood to differentiate definitively between the largely overlapping phenotypes, and in atypical presentations. The two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide. These mutations can be identified by a variety of molecular methods, including PCR with restriction enzyme digestion or direct DNA sequencing. We have developed a single-step, real-time PCR assay in which two detection probes are applied in combination with a single anchor probe at each mutation position. Because the two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide, this approach guarantees optimal differentiation during probe dissociation analysis after amplification. This assay, which is performed on the LightCycler thermocycler, enables the rapid and reliable detection of the two most common FGFR3 mutations associated with ACH (1138G --> A and 1138G --> C; G380R) and HYCH (1620C --> A and 1620 C --> G; N540K) in a single test.