genotyping-by-sequencing
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OBJECTIVES: Direct genotyping of adenovirus or enterovirus from clinical material using polymerase chain reaction (PCR) followed by Sanger sequencing is often difficult due to the presence of multiple virus types in a sample, or due to varying efficacy of PCR amplifying the capsid gene on the background of foreign nucleic acids. Here we present a simple protocol for virus genotyping using massive parallel amplicon sequencing. METHODS: The protocol utilized a set of 16 tailed degenerate primers flanking the seventh hypervariable region of the adenovirus hexon gene and 9 tailed degenerate primers targeted to the proximal portion of the enterovirus VP1 gene. Subsequent addition of dual indices enabled simultaneous sequencing of 384 different samples on an Illumina MiSeq instrument. Downstream bioinformatic analysis was based on remapping to a set of references representative of the presently known repertoire of virus types. RESULTS: After validation with known virus types, the sequencing method was applied on 301 adenovirus-positive samples and 350 enterovirus-positive samples from a longitudinally collected series of stools from 83 children aged 3 to 36 months. We detected 7 different adenovirus types and 27 different enterovirus types. There were 37 (6.2%) samples containing more than one genotype of the same viral genus. At least one dual infection was experienced by 23 of 83 (28%) of the children observed over the 3 years' observation period. CONCLUSIONS: Amplicon sequencing with a multiplex set of degenerate primers seems to be a rapid and reliable technical solution for genotyping of large collections of samples where simultaneous infections with multiple strains can be expected.
- MeSH
- Adenoviridae klasifikace genetika izolace a purifikace MeSH
- adenovirové infekce virologie MeSH
- DNA primery genetika MeSH
- enterovirové infekce virologie MeSH
- Enterovirus klasifikace genetika izolace a purifikace MeSH
- genotyp * MeSH
- genotypizační techniky metody MeSH
- kojenec MeSH
- lidé MeSH
- longitudinální studie MeSH
- předškolní dítě MeSH
- sekvenční analýza DNA metody MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Norsko MeSH
BACKGROUND: Genomic selection (GS) in forestry can substantially reduce the length of breeding cycle and increase gain per unit time through early selection and greater selection intensity, particularly for traits of low heritability and late expression. Affordable next-generation sequencing technologies made it possible to genotype large numbers of trees at a reasonable cost. RESULTS: Genotyping-by-sequencing was used to genotype 1,126 Interior spruce trees representing 25 open-pollinated families planted over three sites in British Columbia, Canada. Four imputation algorithms were compared (mean value (MI), singular value decomposition (SVD), expectation maximization (EM), and a newly derived, family-based k-nearest neighbor (kNN-Fam)). Trees were phenotyped for several yield and wood attributes. Single- and multi-site GS prediction models were developed using the Ridge Regression Best Linear Unbiased Predictor (RR-BLUP) and the Generalized Ridge Regression (GRR) to test different assumption about trait architecture. Finally, using PCA, multi-trait GS prediction models were developed. The EM and kNN-Fam imputation methods were superior for 30 and 60% missing data, respectively. The RR-BLUP GS prediction model produced better accuracies than the GRR indicating that the genetic architecture for these traits is complex. GS prediction accuracies for multi-site were high and better than those of single-sites while multi-site predictability produced the lowest accuracies reflecting type-b genetic correlations and deemed unreliable. The incorporation of genomic information in quantitative genetics analyses produced more realistic heritability estimates as half-sib pedigree tended to inflate the additive genetic variance and subsequently both heritability and gain estimates. Principle component scores as representatives of multi-trait GS prediction models produced surprising results where negatively correlated traits could be concurrently selected for using PCA2 and PCA3. CONCLUSIONS: The application of GS to open-pollinated family testing, the simplest form of tree improvement evaluation methods, was proven to be effective. Prediction accuracies obtained for all traits greatly support the integration of GS in tree breeding. While the within-site GS prediction accuracies were high, the results clearly indicate that single-site GS models ability to predict other sites are unreliable supporting the utilization of multi-site approach. Principle component scores provided an opportunity for the concurrent selection of traits with different phenotypic optima.
- MeSH
- algoritmy MeSH
- dřevo * MeSH
- genomika metody MeSH
- genotypizační techniky * MeSH
- modely genetické MeSH
- sekvenční analýza * MeSH
- šlechtění rostlin metody MeSH
- smrk genetika růst a vývoj MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Echinococcus granulosus (Batsch, 1786), a cestode of the Teniidae family, causes human cystic echinococcosis (CE) also known as hydatid disease. Echinococcus granulosus sensu lato includes the G1, G3, G4, G5, G6/7 and G8/10 genotypes which are known to cause human CE. This study aimed to differentiate genotypes of E. granulosus s.l. complex by employing EmsB, a tandemly repeated multilocus microsatellite, using next-generation sequencing (MIC-NGS). Human and animal histopathology-confirmed hydatid cyst tissue samples and reference DNA samples of E. granulosus G1, G3, G4, G5, G6/7 and G10 underwent MIC-NGS assay with custom primers amplifying a 151 bp EmsB DNA fragment. NGS data were analysed using online Galaxy analysis pipeline, a phylogenetic tree was constructed by MEGA software, and haplotype networking was performed with PopArt 1.7. All sixty samples (49 from animals and 11 from humans) included were successfully identified and genotyped with a 100 % success rate. The study showed improved discrimination power to distinguish all study samples including closely related E. granulosus s.s. genotypes G1-G3. The maximum likelihood tree reaffirmed the monophyly of E. granulosus s.l. The median-joining haplotype networking revealed 12 distinct haplotypes. In conclusion, MIC-NGS assay was shown to be sensitive, specific and simple to apply to clinical samples offering a powerful discriminatory tool for the genotyping of E. granulosus s.l.
- MeSH
- Echinococcus granulosus * genetika MeSH
- echinokokóza * veterinární parazitologie MeSH
- fylogeneze MeSH
- genotyp * MeSH
- genotypizační techniky veterinární MeSH
- lidé MeSH
- mikrosatelitní repetice * MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Tuberculosis (TB) is considered one of the most serious infectious diseases worldwide. Effective control of tuberculosis infection involves multiple steps, such as reliable detection, treatment, an epidemiological control as a part of case management, and further surveillance and monitoring of TB spread in the human population. Due to the accelerating advances in molecular biology, especially in DNA sequencing, in the past decade, the application of these methods has become crucial for TB evolution studies, differentiation of Mycobacterium tuberculosis genotypes, and their distribution. Currently, several molecular genetic methods are available. The oldest typing methods (e.g., IS6110-RFLP, spoligotyping, and MIRU-VNTR) can discover the chain of transmission to the patient. Currently, whole genome sequencing facilitates is furthermore able to identify the source of infection, the transmission trays among individuals sharing the same isolate, as well as determination of the TB evolution and its resistance to antituberculotic agents. It is obvious that this technique will become a new gold standard in genotyping methods in tuberculosis molecular epidemiological studies. In this article, molecular genetic typing methods with a special focus on whole genome sequencing and data management are reviewed.
- MeSH
- bakteriální léková rezistence genetika MeSH
- fylogeografie MeSH
- genom bakteriální * MeSH
- genotyp MeSH
- lidé MeSH
- molekulární epidemiologie normy MeSH
- molekulární typizace metody normy MeSH
- Mycobacterium tuberculosis klasifikace genetika MeSH
- sekvenování celého genomu * MeSH
- tuberkulóza diagnóza epidemiologie mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
PURPOSE: A new high-resolution next-generation sequencing (NGS)-based method was established to type closely related European type II Toxoplasma gondii strains. METHODS: T. gondii field isolates were collected from different parts of Europe and assessed by whole genome sequencing (WGS). In comparison to ME49 (a type II reference strain), highly polymorphic regions (HPRs) were identified, showing a considerable number of single nucleotide polymorphisms (SNPs). After confirmation by Sanger sequencing, 18 HPRs were used to design a primer panel for multiplex PCR to establish a multilocus Ion AmpliSeq typing method. Toxoplasma gondii isolates and T. gondii present in clinical samples were typed with the new method. The sensitivity of the method was tested with serially diluted reference DNA samples. RESULTS: Among type II specimens, the method could differentiate the same number of haplotypes as the reference standard, microsatellite (MS) typing. Passages of the same isolates and specimens originating from abortion outbreaks were identified as identical. In addition, seven different genotypes, two atypical and two recombinant specimens were clearly distinguished from each other by the method. Furthermore, almost all SNPs detected by the Ion AmpliSeq method corresponded to those expected based on WGS. By testing serially diluted DNA samples, the method exhibited a similar analytical sensitivity as MS typing. CONCLUSION: The new method can distinguish different T. gondii genotypes and detect intra-genotype variability among European type II T. gondii strains. Furthermore, with WGS data additional target regions can be added to the method to potentially increase typing resolution.
- MeSH
- genetická variace MeSH
- genotyp MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- protozoální DNA genetika MeSH
- těhotenství MeSH
- Toxoplasma * genetika MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Bifidobacteria are widely known for their probiotic potential; however, little is known regarding the ecological significance and potential probiotic effects of the phylogenetically related 'scardovial' genera (Aeriscardovia, Alloscardovia, Bombiscardovia, Galliscardovia, Neoscardovia, Parascardovia, Pseudoscardovia and Scardovia) and Gardnerella classified with bifidobacteria within the Bifidobacteriaceae family. Accurate classification and genotyping of bacteria using certain housekeeping genes is possible, whilst current phylogenomic analyses allow for extremely precise classification. Studies of applicable genetic markers may provide results comparable to those obtained from phylogenomic analyses of the family Bifidobacteriaceae. Segments of the glyS (624 nucleotides), pheS (555 nucleotides), rpsA (630 nucleotides), and rpsB (432 nucleotides) genes and their concatenated sequence were explored. The mean glyS, pheS, rpsB and rpsA gene sequence similarities calculated for Bifidobacterium taxa were 84.8, 85.2, 90.2 and 86.8%, respectively. Interestingly, the average value of the Average Nucleotide Identity among 67 type strains of the family Bifidobacteriaceae (84.70%) calculated based on values published recently was in agreement with the average pairwise similarity (84.6%) among 75 type strains of Bifidobacteriaceae family computed in this study using the concatenated sequences of four gene fragments. Similar to phylogenomic analyses, several gene sequence and phylogenetic analyses revealed that concatenated gene regions allow for classification of Bifidobacteriaceae strains into particular phylogenetic clusters and groups. Phylogeny reconstructed from the concatenated sequences assisted in defining two novel phylogenetic groups, the Bifidobacterium psychraerophilum group consisting of B. psychraerophilum, Bifidobacterium crudilactis and Bifidobacterium aquikefiri species and the Bifidobacterium bombi group consisting of B. bombi, Bifidobacterium bohemicum and Bifidobacterium commune.
Spot blotch (SB) caused by Bipolaris sorokiniana and powdery mildew (PM) caused by Blumeria graminis f. sp. hordei are two important diseases of barley. To map genetic loci controlling susceptibility and resistance to these diseases, a mapping population consisting of 138 recombinant inbred lines (RILs) was developed from the cross between Bowman and ND5883. A genetic map was constructed for the population with 852 unique single nucleotide polymorphism markers generated by sequencing-based genotyping. Bowman and ND5883 showed distinct infection responses at the seedling stage to two isolates (ND90Pr and ND85F) of Bipolaris sorokiniana and one isolate (Race I) of Blumeria graminis f. sp. hordei. Genetic analysis of the RILs revealed that one major gene (Scs6) controls susceptibility to Bipolaris sorokiniana isolate ND90Pr, and another major gene (Mla8) confers resistance to Blumeria graminis f. sp. hordei isolate Race I, respectively. Scs6 was mapped on chromosome 1H of Bowman, as previously reported. Mla8 was also mapped to the short arm of 1H, which was tightly linked but not allelic to the Rcs6/Scs6 locus. Quantitative trait locus (QTL) analysis identified two QTLs, QSbs-1H-P1 and QSbs-7H-P1, responsible for susceptibility to spot blotch caused by Bipolaris sorokiniana isolate ND85F in ND5883, which are located on chromosome 1H and 7H, respectively. QSbs-7H-P1 was mapped to the same region as Rcs5, whereas QSbs-1H-P1 may represent a novel allele conferring seedling stage susceptibility to isolate ND85F. Identification and molecular mapping of the loci for SB susceptibility and PM resistance will facilitate development of barley cultivars with resistance to the diseases.
With the rise of next-generation sequencing methods, it has become increasingly possible to obtain genomewide sequence data even for nonmodel species. Such data are often used for the development of single nucleotide polymorphism (SNP) markers, which can subsequently be screened in a larger population sample using a variety of genotyping techniques. Many of these techniques require appropriate locus-specific PCR and genotyping primers. Currently, there is no publicly available software for the automated design of suitable PCR and genotyping primers from next-generation sequence data. Here we present a pipeline called Scrimer that automates multiple steps, including adaptor removal, read mapping, selection of SNPs and multiple primer design from transcriptome data. The designed primers can be used in conjunction with several widely used genotyping methods such as SNaPshot or MALDI-TOF genotyping. Scrimer is composed of several reusable modules and an interactive bash workflow that connects these modules. Even the basic steps are presented, so the workflow can be executed in a step-by-step manner. The use of standard formats throughout the pipeline allows data from various sources to be plugged in, as well as easy inspection of intermediate results with visualization tools of the user's choice.
Cyprinids are the most important group of farmed fish globally in terms of production volume, with common carp (Cyprinus carpio) being one of the most valuable species of the group. The use of modern selective breeding methods in carp is at a formative stage, implying a large scope for genetic improvement of key production traits. In the current study, a population of 1,425 carp juveniles, originating from a partial factorial cross between 40 sires and 20 dams, was used for investigating the potential of genomic selection (GS) for juvenile growth, an exemplar polygenic production trait. RAD sequencing was used to identify and genotype SNP markers for subsequent parentage assignment, construction of a medium density genetic map (12,311 SNPs), genome-wide association study (GWAS), and testing of GS. A moderate heritability was estimated for body length of carp at 120 days (as a proxy of juvenile growth) of 0.33 (s.e. 0.05). No genome-wide significant QTL was identified using a single marker GWAS approach. Genomic prediction of breeding values outperformed pedigree-based prediction, resulting in 18% improvement in prediction accuracy. The impact of reduced SNP densities on prediction accuracy was tested by varying minor allele frequency (MAF) thresholds, with no drop in prediction accuracy until the MAF threshold is set <0.3 (2,744 SNPs). These results point to the potential for GS to improve economically important traits in common carp breeding programs.
- Publikační typ
- časopisecké články MeSH
AIM: The primary goal was to determine the yield of next-generation sequencing (NGS) epilepsy gene panels used for epilepsy etiology diagnosing using a multidisciplinary approach and to demonstrate the importance of genotype-phenotype correlations. The secondary goal was to evaluate the application of precision medicine in selected patients. METHODS: This single-center retrospective study included a total of 175 patients (95 males and 80 females) aged 0-19 years. They were examined between 2015 and 2020 using an NGS epilepsy gene panel (270 genes). A bioinformatic analysis was performed including copy number variation identification. Thorough genotype-phenotype correlation was performed. RESULTS: Out of 175 patients, described pathogenic variants or novel variants with clear pathogenic impact were identified in 30 patients (17.14%). Genotype-phenotype correlations and parental DNA analysis were performed, and genetic diagnosis was confirmed on the basis of the results in another 16 out of 175 patients (9.14%). The diagnostic yield of our study increased from 30 to 46 patients (by 53.33%) by the precise genotype-phenotype correlation. INTERPRETATION: We emphasize a complex genotype-phenotype correlation and a multidisciplinary approach in evaluating the results of the NGS epilepsy gene panel, which enables the most accurate genetic diagnosis and correct interpretation of results.
- MeSH
- epilepsie * diagnóza genetika MeSH
- fenotyp MeSH
- genetické asociační studie MeSH
- genetické testování metody MeSH
- lidé MeSH
- mutace MeSH
- retrospektivní studie MeSH
- variabilita počtu kopií segmentů DNA * MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
