Pseudomonas aeruginosa is a rod-shaped Gram-negative bacterium which is notably known as a pathogen in humans, animals, and plants. Infections caused by P. aeruginosa especially in hospitalized patients are often life-threatening and rapidly increasing worldwide throughout the years. Recently, multidrug-resistant P. aeruginosa has taken a toll on humans' health due to the inefficiency of antimicrobial agents. Therefore, the rapid and advanced diagnostic techniques to accurately detect this bacterium particularly in clinical samples are indeed necessary to ensure timely and effective treatments and to prevent outbreaks. This review aims to discuss most recent of state-of-the-art molecular diagnostic techniques enabling fast and accurate detection and identification of P. aeruginosa based on well-developed genotyping techniques, e.g., polymerase chain reaction, pulse-field gel electrophoresis, and next generation sequencing. The advantages and limitations of each of the methods are also reviewed.

Polycystic ovary syndrome (PCOS), the commonest endocrine disorder affecting young women, appears to be a multigenic trait with contributing genes being unclear. Hence, analysis of polymorphisms in multiple candidate genes is required. Currently available genotyping methods are expensive, time-consuming with limited analytical sensitivity.

Background: Graft-derived cell-free DNA (gdcfDNA) analysis has shown promise as a non-invasive tool for monitoring organ health following solid organ transplantation. A number of gdcfDNA analysis techniques have been described; however, the majority rely on sequencing or prior genotyping to detect donor-recipient mis-matched genetic polymorphisms. Differentially methylated regions of DNA can be used to identify the tissue-of-origin of cell-free DNA (cfDNA) fragments. In this study, we aimed to directly compare the performance of gdcfDNA monitoring using graft-specific DNA methylation analysis and donor-recipient genotyping techniques in a pilot cohort of clinical samples from patients post-liver transplantation. Results: 7 patients were recruited prior to LT, 3 developed early, biopsy-proven TCMR in the first 6 weeks post-LT. gdcfDNA was successfully quantified in all samples using both approaches. There was a high level of technical correlation between results using the two techniques (Spearman testing, rs = 0.87, p < 0.0001). gdcfDNA levels quantified using the genotyping approach were significantly greater across all timepoints in comparison to the tissue-specific DNA methylation-based approach: e.g., day 1 post-LT median 31,350 copies/mL (IQR 6731-64,058) vs. 4133 copies/mL (IQR 1100-8422), respectively. Qualitative trends in gdcfDNA levels for each patient were concordant between the two assays. Acute TCMR was preceded by significant elevations in gdcfDNA as quantified by both techniques. Elevations in gdcfDNA, using both techniques, were suggestive of TCMR in this pilot study with a 6- and 3-day lead-time prior to histological diagnosis in patients 1 and 2. Conclusions: Both the graft-specific methylation and genotyping techniques successfully quantified gdcfDNA in patients post-LT with statistically significant concordance. A direct comparison of these two techniques is not only important from a technical perspective for orthogonal validation, but significantly adds weight to the evidence that gdcfDNA monitoring reflects the underlying biology. Both techniques identified LT recipients who developed acute TCMR, with several days lead-time in comparison to conventional diagnostic workflows. Whilst the two assays performed comparably, gdcfDNA monitoring based on graft-specific DNA methylation patterns in cfDNA offers major practical advantages over the donor-recipient genotyping, and hence enhances the potential to translate this emerging technology into clinical practice.

Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI), GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan); genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/μL in cDNA sample, corresponding to 101-104 copies/mL-sewage, 105-108 copies/g-human feces, and 102-105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

In tomato, resistance to Tomato spotted wilt virus (TSWV) is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB) mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM) assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI) and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies) and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke) and summer (tomato) crops, in the same cultivated areas of Southern Italy.

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo's sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p < 0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak's height was higher and this peak's presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p < 0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

The genotyping of mother-father-child trios is a very useful tool in disease association studies, as trios eliminate population stratification effects and increase the accuracy of haplotype inference. Unfortunately, the use of trios for association studies may reduce power, since it requires the genotyping of three individuals where only four independent haplotypes are involved. We describe here a method for genotyping a trio using two DNA pools, thus reducing the cost of genotyping trios to that of genotyping two individuals. Furthermore, we present extensions to the method that exploit the linkage disequilibrium structure to compensate for missing data and genotyping errors. We evaluated our method on trios from CEPH pedigree 66 of the Coriell Institute. We demonstrate that the error rates in the genotype calls of the proposed protocol are comparable to those of standard genotyping techniques, although the cost is reduced considerably. The approach described is generic and it can be applied to any genotyping platform that achieves a reasonable precision of allele frequency estimates from pools of two individuals. Using this approach, future trio-based association studies may be able to increase the sample size by 50% for the same cost and thereby increase the power to detect associations.

Historically, the laboratory mouse has been the mammalian species of choice for studying gene function and for modeling diseases in humans. This was mainly due to their availability from mouse fanciers. In addition, their short generation time, small size, and minimal food consumption compared to that of larger mammals were definite advantages. This led to the establishment of large hubs for the development of genetically modified mouse models, such as the Jackson Laboratory. Initial research into inbred mouse strains in the early 1900s revolved around coat color genetics and cancer studies, but gene targeting in embryonic stem cells and the introduction of transgenes through pronuclear injection of a mouse zygote, along with current clustered regularly interspaced short palindromic repeat (CRISPR) RNA gene editing, have allowed easy manipulation of the mouse genome. Originally, to distribute a mouse model to other facilities, standard methods had to be developed to ensure that each modified mouse trait could be consistently identified no matter which laboratory requested it. The task of establishing uniform protocols became easier with the development of the polymerase chain reaction (PCR). This chapter will provide guidelines for identifying genetically modified mouse models, mainly using endpoint PCR. In addition, we will discuss strategies to identify genetically modified mouse models that have been established using newer gene-editing technology such as CRISPR. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Digestion with proteinase K followed by purification of genomic DNA using phenol/chloroform Alternate Protocol: Digestion with proteinase K followed by crude isopropanol extraction of genomic DNA for tail biopsy and ear punch samples Basic Protocol 2: Purification of genomic DNA using a semi-automated system Basic Protocol 3: Purification of genomic DNA from semen, blood, or buccal swabs Basic Protocol 4: Purification of genomic DNA from mouse blastocysts to assess CRISPR gene editing Basic Protocol 5: Routine endpoint-PCR-based genotyping using DNA polymerase and thermal cycler Basic Protocol 6: T7E1/Surveyor assays to detect insertion or deletions following CRISPR editing Basic Protocol 7: Detecting off-target mutations following CRISPR editing Basic Protocol 8: Detecting genomic sequence deletion after CRISPR editing using a pair of guide RNAs Basic Protocol 9: Detecting gene knock-in events following CRISPR editing Basic Protocol 10: Screening of conditional knockout floxed mice.

Latest genotyping solutions allow for rapid testing of more than two million markers in one experiment. Fully automated instruments such as Affymetrix GeneTitan enable processing of large numbers of samples in a truly high-throughput manner. In concert with solutions like Axiom, fully customizable array plates can now utilize automated workflows that can leverage multi-channel instrumentation like the GeneTitan. With the growing size of raw data output, the serial computational architecture of the software, typically distributed by the vendors on turnkey desktop solutions for quality control and genotype calling, becomes legacy rather than an advantage. Advanced software techniques provide power, flexibility, and can be deployed in an HPC environment, but become technically inconvenient for biologists to use. Here we present a pipeline that uses Galaxy as a mechanism to lower the barrier for complex analysis, and increase efficiency by leveraging high-throughput computing.

Microsatellite markers are simple sequence repeats within the mammalian genome that can be used for identifying disease loci, mapping genes of interest as well as studying segregation patterns related to meiotic nondisjunction. Different strains of mice have variable CA repeat lengths and PCR based methods can be used to identify them, thus allowing for specific genotypes to be assigned. Molecular genotyping offers such identification at any developmental stage, which allows for a broad range of anomalies to be studied. We studied chromosomal segregation in relation to nondisjunction in early-gestation mouse embryos using molecular genotyping. Information on the parental origin as well as the number of chromosomes a given progeny carried was obtained in our analysis.

Next-generation high-throughput DNA sequencing techniques are opening fascinating opportunities in the life sciences. Novel fields and applications in biology and medicine are becoming a reality, beyond the genomic sequencing which was original development goal and application. Serving as examples are: personal genomics with detailed analysis of individual genome stretches; precise analysis of RNA transcripts for gene expression, surpassing and replacing in several respects analysis by various microarray platforms, for instance in reliable and precise quantification of transcripts and as a tool for identification and analysis of DNA regions interacting with regulatory proteins in functional regulation of gene expression. The next-generation sequencing technologies offer novel and rapid ways for genome-wide characterisation and profiling of mRNAs, small RNAs, transcription factor regions, structure of chromatin and DNA methylation patterns, microbiology and metagenomics. In this article, development of commercial sequencing devices is reviewed and some European contributions to the field are mentioned. Presently commercially available very high-throughput DNA sequencing platforms, as well as techniques under development, are described and their applications in bio-medical fields discussed.

Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

It has recently been demonstrated that organism identifications can be recovered from mass spectra using various methods including base-specific fragmentation of nucleic acids. Because mass spectrometry is extremely rapid and widely available such techniques offer significant advantages in some applications. A key element in favor of mass spectrometric analysis of RNA fragmentation patterns is that a reference database for analysis of the results can be generated from sequence information. In contrast to hybridization approaches, the genetic affinity of any unknown isolate can in principle be determined within the context of all previously sequenced 16S rRNAs without prior knowledge of what the organism is. In contrast to the original RNase T1 cataloging method, when digestion products are analyzed by mass spectrometry, products with the same base composition cannot be distinguished. Hence, it is possible that organisms that are not closely related (having different underlying sequences) might be falsely identified by mass spectral coincidence. We present a convenient spectral coincidence function for expressing the degree of similarity (or distance) between any two mass-spectra. Trees constructed using this function are consistent with those produced by direct comparison of primary sequences, demonstrating that the inherent degeneracy in mass spectrometric analysis of RNA fragments does not preclude correct organism identification.

Enteroviruses (EV) with different genotypes cause diverse infectious diseases in humans and mammals. A correct EV typing result is crucial for effective medical treatment and disease control; however, the emergence of novel viral strains has impaired the performance of available diagnostic tools. Here, we present a web-based tool, named EVIDENCE (EnteroVirus In DEep conception, http://symbiont.iis.sinica.edu.tw/evidence), for EV genotyping and recombination detection. We introduce the idea of using mixed-ranking scores to evaluate the fitness of prototypes based on relatedness and on the genome regions of interest. Using phylogenetic methods, the most possible genotype is determined based on the closest neighbor among the selected references. To detect possible recombination events, EVIDENCE calculates the sequence distance and phylogenetic relationship among sequences of all sliding windows scanning over the whole genome. Detected recombination events are plotted in an interactive figure for viewing of fine details. In addition, all EV sequences available in GenBank were collected and revised using the latest classification and nomenclature of EV in EVIDENCE. These sequences are built into the database and are retrieved in an indexed catalog, or can be searched for by keywords or by sequence similarity. EVIDENCE is the first web-based tool containing pipelines for genotyping and recombination detection, with updated, built-in, and complete reference sequences to improve sensitivity and specificity. The use of EVIDENCE can accelerate genotype identification, aiding clinical diagnosis and enhancing our understanding of EV evolution.

The molecular typing of Mycobacterium bovis, which causes bovine tuberculosis, can be accomplished by combining different polymorphic markers, contributing to its epidemiological investigation. Multispacer sequence typing (MST) is a sequencing-based method that employs intergenic regions susceptible to higher mutation rates given the low selection pressure. It has been applied to M. tuberculosis, but not to M. bovis. The aim of this study was to evaluate a MST for M. bovis. A total of 58 strains isolated from tissues with lesions suggestive of bovine tuberculosis, coming from cattle herds in six Brazilian states and four standard samples of M. bovis were typified employing the MST technique. Fourteen intergenic regions were used, and four types of genetic events were reported: single nucleotide mutation (SNP), insertion, deletion, and tandem repeat (TR). Seven loci were chosen for typing. Twenty-eight type sequences (ST) were identified, indicating type sequences (ST) were identified, indicating a 92.9% HGDI (Hunter Gaston Discriminatory Index). The data were used to analyze the evolutionary patterns of these isolates and correlate them to phylogeographic lineages based on the formation of clonal complexes generated from eBURST software. Later, we associated the MST with spoligotyping technique, currently considered the gold standard for classification of M. bovis. The results support the MST as an alternative method for genotyping of M. bovis. The method has the advantage of sequencing and the availability of sequences analyzed in public databases, which can be used by professionals around the world as a tool for further analysis. This was the first study to identify the variability of isolates of M. bovis by the MST method.

Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The 'gold standard' genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST.

Application of next generation sequencing for large scale genotyping in livestock is limited by high costs and challenging data analysis process. However, available restriction enzyme-based enrichment techniques like e.g. genotyping-by-sequencing (GBS) are promising tools allowing reduction of financial outlies by a high sample multiplexing and narrowing down the sequenced genome areas to the randomly distributed read tags. In this study, we tested the performance of standard, PstI endonuclease-adapted GBS protocol for population genetics in cattle, horse and sheep with application of different, including low-depth sequencing setups. It was found that the detected SNPs display desirable polymorphism parameters and are evenly scattered across the whole genome including gene coding regions. It was also shown that the SNPs can be successfully applied in population genetics, revealing the genetic differentiation of the studied breeds. The GBS approach represents a cost-effective alternative to existing genotyping methods which may find adoption in various research applications.

The emerging global infectious COVID-19 disease by novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents critical threats to global public health and the economy since it was identified in late December 2019 in China. The virus has gone through various pathways of evolution. To understand the evolution and transmission of SARS-CoV-2, genotyping of virus isolates is of great importance. This study presents an accurate method for effectively genotyping SARS-CoV-2 viruses using complete genomes. The method employs the multiple sequence alignments of the genome isolates with the SARS-CoV-2 reference genome. The single-nucleotide polymorphism (SNP) genotypes are then measured by Jaccard distances to track the relationship of virus isolates. The genotyping analysis of SARS-CoV-2 isolates from the globe reveals that specific multiple mutations are the predominated mutation type during the current epidemic. The proposed method serves an effective tool for monitoring and tracking the epidemic of pathogenic viruses in their global and local genetic variations. The genotyping analysis shows that the genes encoding the S proteins and RNA polymerase, RNA primase, and nucleoprotein, undergo frequent mutations. These mutations are critical for vaccine development in disease control.

Genotyping microarrays are an important resource for genetic mapping, population genetics, and monitoring of the genetic integrity of laboratory stocks. We have developed the third generation of the Mouse Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the content of GigaMUGA is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations, and for substrain-level identification of laboratory mice. In addition to 141,090 single nucleotide polymorphism probes, GigaMUGA contains 2006 probes for copy number concentrated in structurally polymorphic regions of the mouse genome. The performance of the array is characterized in a set of 500 high-quality reference samples spanning laboratory inbred strains, recombinant inbred lines, outbred stocks, and wild-caught mice. GigaMUGA is highly informative across a wide range of genetically diverse samples, from laboratory substrains to other Mus species. In addition to describing the content and performance of the array, we provide detailed probe-level annotation and recommendations for quality control.

Because of their abundance and their amenability to high-throughput genotyping techniques, Single Nucleotide Polymorphisms (SNPs) are powerful tools for efficient genetics and genomics studies, including characterization of genetic resources, genome-wide association studies and genomic selection. In wheat, most of the previous SNP discovery initiatives targeted the coding fraction, leaving almost 98% of the wheat genome largely unexploited. Here we report on the use of whole-genome resequencing data from eight wheat lines to mine for SNPs in the genic, the repetitive and non-repetitive intergenic fractions of the wheat genome. Eventually, we identified 3.3 million SNPs, 49% being located on the B-genome, 41% on the A-genome and 10% on the D-genome. We also describe the development of the TaBW280K high-throughput genotyping array containing 280,226 SNPs. Performance of this chip was examined by genotyping a set of 96 wheat accessions representing the worldwide diversity. Sixty-nine percent of the SNPs can be efficiently scored, half of them showing a diploid-like clustering. The TaBW280K was proven to be a very efficient tool for diversity analyses, as well as for breeding as it can discriminate between closely related elite varieties. Finally, the TaBW280K array was used to genotype a population derived from a cross between Chinese Spring and Renan, leading to the construction a dense genetic map comprising 83,721 markers. The results described here will provide the wheat community with powerful tools for both basic and applied research.