High-throughput DNA sequencing techniques enable time- and cost-effective sequencing of large portions of the genome. Instead of sequencing and annotating whole genomes, many phylogenetic studies focus sequencing effort on large sets of pre-selected loci, which further reduces costs and bioinformatic challenges while increasing coverage. One common approach that enriches loci before sequencing is often referred to as target sequence capture. This technique has been shown to be applicable to phylogenetic studies of greatly varying evolutionary depth. Moreover, it has proven to produce powerful, large multi-locus DNA sequence datasets suitable for phylogenetic analyses. However, target capture requires careful considerations, which may greatly affect the success of experiments. Here we provide a simple flowchart for designing phylogenomic target capture experiments. We discuss necessary decisions from the identification of target loci to the final bioinformatic processing of sequence data. We outline challenges and solutions related to the taxonomic scope, sample quality, and available genomic resources of target capture projects. We hope this review will serve as a useful roadmap for designing and carrying out successful phylogenetic target capture studies.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The North American ecological species Daphniapulicaria and Daphniapulex are thought to have diverged from a common ancestor by adaptation to sympatric but ecologically distinct lake and pond habitats respectively. Based on mtDNA relationships, European D. pulicaria is considered a different species only distantly related to its North American counterpart, but both species share a lactate dehydrogenase (Ldh) allele F supposedly involved in lake adaptation in North America, and the same allele is also carried by the related Holarctic Daphniatenebrosa. The correct inference of the species' ancestral relationships is therefore critical for understanding the origin of their adaptive divergence. Our species tree inferred from unlinked nuclear loci for D. pulicaria and D. pulex resolved the European and North American D. pulicaria as sister clades, and we argue that the discordant mtDNA gene tree is best explained by capture of D. pulex mtDNA by D. pulicaria in North America. The Ldh gene tree shows that F-class alleles in D. pulicaria and D. tenebrosa are due to common descent (as opposed to introgression), with D. tenebrosa alleles paraphyletic with respect to D. pulicaria alleles. That D. tenebrosa still segregates the ancestral and derived amino acids at the two sites distinguishing the pond and lake alleles suggests that D. pulicaria inherited the derived states from the D. tenebrosa ancestry. Our results suggest that some adaptations restricting the gene flow between D. pulicaria and D. pulex might have evolved in response to selection in ancestral environments rather than in the species' current sympatric habitats. The Arctic (D. tenebrosa) populations are likely to provide important clues about these issues.

• MeSH
• Daphnia klasifikace   genetika   MeSH
• druhová specificita MeSH
• fylogeneze MeSH
• fylogeografie MeSH
• mitochondriální DNA chemie   MeSH
• rekombinace genetická MeSH
• sekvenční analýza DNA MeSH
• tok genů MeSH
• vznik druhů (genetika) * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Retroelements (REs) occupy a significant part of all eukaryotic genomes including humans. The majority of retroelements in the human genome are inactive and unable to retrotranspose. Dozens of active copies are repressed in most normal tissues by various cellular mechanisms. These copies can become active in normal germline and brain tissues or in cancer, leading to new retroposition events. The consequences of such events and their role in normal cell functioning and carcinogenesis are not yet fully understood. If new insertions occur in a small portion of cells they can be found only with the use of specific methods based on RE enrichment and high-throughput sequencing. The downside of the high sensitivity of such methods is the presence of various artifacts imitating real insertions, which in many cases cannot be validated due to lack of the initial template DNA. For this reason, adequate assessment of rare (< 1%) subclonal cancer specific RE insertions is complicated. RESULTS: Here we describe a new copy-capture technique which we implemented in a method called SeqURE for Sequencing Unknown of Retroposition Events that allows for efficient and reliable identification of new genomic RE insertions. The method is based on the capture of copies of target molecules (copy-capture), selective amplification and sequencing of genomic regions adjacent to active RE insertions from both sides. Importantly, the template genomic DNA remains intact and can be used for validation experiments. In addition, we applied a novel system for testing method sensitivity and precisely showed the ability of the developed method to reliably detect insertions present in 1 out of 100 cells and a substantial portion of insertions present in 1 out of 1000 cells. Using advantages of the method we showed the absence of somatic Alu insertions in colorectal cancer samples bearing tumor-specific L1HS insertions. CONCLUSIONS: This study presents the first description and implementation of the copy-capture technique and provides the first methodological basis for the quantitative assessment of RE insertions present in a small portion of cells.

• Publikační typ
• časopisecké články MeSH

Chromosomal inversions occur in natural populations of many species, and may underlie reproductive isolation and local adaptation. Traditional methods of inversion discovery are labor-intensive and lack sensitivity. Here, we report the use of three-dimensional contact probabilities between genomic loci as assayed by chromosome-conformation capture sequencing (Hi-C) to detect multi-megabase polymorphic inversions in four barley genotypes. Inversions are validated by fluorescence in situ hybridization and Bionano optical mapping. We propose Hi-C as a generally applicable method for inversion discovery in natural populations.

• MeSH
• chromozomální inverze genetika   MeSH
• chromozomy rostlin genetika   MeSH
• genom rostlinný genetika   MeSH
• genotyp MeSH
• hybridizace in situ fluorescenční MeSH
• ječmen (rod) genetika   MeSH
• mapování chromozomů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In the carnivorous plant Venus flytrap (Dionaea muscipula), the sequence of events after prey capture resembles the well-known plant defence signalling pathway in response to pathogen or herbivore attack. Here, we used wounding to mimic prey capture to show the similarities and differences between botanical carnivory and plant defence mechanisms. We monitored movement, electrical signalling, jasmonate accumulation and digestive enzyme secretion in local and distal (systemic) traps in response to prey capture, the mechanical stimulation of trigger hairs and wounding. The Venus flytrap cannot discriminate between wounding and mechanical trigger hair stimulation. Both induced the same action potentials, rapid trap closure, hermetic trap sealing, the accumulation of jasmonic acid (JA) and its isoleucine conjugate (JA-Ile), and the secretion of proteases (aspartic and cysteine proteases), phosphatases and type I chitinase. The jasmonate accumulation and enzyme secretion were confined to the local traps, to which the stimulus was applied, which correlates with the propagation of electrical signals and the absence of a systemic response in the Venus flytrap. In contrast to plant defence mechanisms, the absence of a systemic response in carnivorous plant may represent a resource-saving strategy. During prey capture, it could be quite expensive to produce digestive enzymes in the traps on the plant without prey.

• MeSH
• chitinasy metabolismus   MeSH
• cyklopentany metabolismus   MeSH
• Droseraceae fyziologie   MeSH
• enzymy metabolismus   MeSH
• hmyz MeSH
• oxylipiny metabolismus   MeSH
• rostlinné proteiny metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Genomic selection (GS) can offer unprecedented gains, in terms of cost efficiency and generation turnover, to forest tree selective breeding; especially for late expressing and low heritability traits. Here, we used: 1) exome capture as a genotyping platform for 1372 Douglas-fir trees representing 37 full-sib families growing on three sites in British Columbia, Canada and 2) height growth and wood density (EBVs), and deregressed estimated breeding values (DEBVs) as phenotypes. Representing models with (EBVs) and without (DEBVs) pedigree structure. Ridge regression best linear unbiased predictor (RR-BLUP) and generalized ridge regression (GRR) were used to assess their predictive accuracies over space (within site, cross-sites, multi-site, and multi-site to single site) and time (age-age/ trait-trait). RESULTS: The RR-BLUP and GRR models produced similar predictive accuracies across the studied traits. Within-site GS prediction accuracies with models trained on EBVs were high (RR-BLUP: 0.79-0.91 and GRR: 0.80-0.91), and were generally similar to the multi-site (RR-BLUP: 0.83-0.91, GRR: 0.83-0.91) and multi-site to single-site predictive accuracies (RR-BLUP: 0.79-0.92, GRR: 0.79-0.92). Cross-site predictions were surprisingly high, with predictive accuracies within a similar range (RR-BLUP: 0.79-0.92, GRR: 0.78-0.91). Height at 12 years was deemed the earliest acceptable age at which accurate predictions can be made concerning future height (age-age) and wood density (trait-trait). Using DEBVs reduced the accuracies of all cross-validation procedures dramatically, indicating that the models were tracking pedigree (family means), rather than marker-QTL LD. CONCLUSIONS: While GS models' prediction accuracies were high, the main driving force was the pedigree tracking rather than LD. It is likely that many more markers are needed to increase the chance of capturing the LD between causal genes and markers.

• MeSH
• dřevo chemie   genetika   MeSH
• exom * MeSH
• genomika MeSH
• genotyp MeSH
• lineární modely MeSH
• lokus kvantitativního znaku MeSH
• modely genetické * MeSH
• Pseudotsuga genetika   růst a vývoj   MeSH
• selekce (genetika) * MeSH
• šlechtění rostlin * MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH

The arms race between specialist predators and their prey has resulted in the evolution of a variety of specific adaptations. In venomous predators, this can include venom composition, particularly if predators are specialized on dangerous prey. Here, we performed an integrative study using six species of highly specialized ant-eating spiders of the genus Zodarion to investigate their phylogeny, realized trophic niche, efficacy in the capture of various ant species and venom composition. Data on natural diet obtained by next-generation sequencing and field observations showed that the six Zodarion species exploit different ant species. Their phylogeny, based on mitochondrial and nuclear genes, correlated with the composition of their natural prey, indicating that closely related Zodarion species specialize on similar ant species. Prey-capture parameters differed among Zodarion species suggesting prey-specific efficacy. Similarly, the venom profiles of both low and high molecular compounds differed among species. Only the profiles of low molecular compounds were correlated with capture efficacy parameters, suggesting that the venom of Zodarion spiders contains prey-specific components. Our study suggests that Iberian Zodarion spiders are specialized on particular ant species.

• MeSH
• ekosystém * MeSH
• Formicidae MeSH
• fylogeneze MeSH
• pavoučí jedy analýza   MeSH
• pavouci fyziologie   MeSH
• predátorské chování MeSH
• stravovací zvyklosti * MeSH
• sympatrie fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cílem projektu je zavedení a validace molekulárně genetické diagnostiky neuromuskulárních chorob s využitím technologie "Sequence capture (SeqCap) and Targeted resequencig (TR)". SeqCap je metoda, která umožňuje obohacení vybraných úseků genomové DNA (např. exonů genů asociovaných s neuromuskulárními nemocemi) v jednom kroku, a ve spojení s "next-generation" sekvenováním tak vytváří předpoklady pro rychlou a cenově příznivou analýzu rozsáhlých oblastí genomu.; Objective of the project is the design, implementation, and validation of molecular genetic diagnostic of neuromuscular disorders by using of Sequence capture (SeqCap) and Targeted resequencig (TR). SeqCap is a method which enables the capture and enrichment of selected regions of genomic DNA (for example exons of genes associated with neuromuscular disorders) in one step and with association with next-generation sequencing creates conditions for fast and cost-effective analysis of extensive areas of the genome.

• MeSH
• diagnostické techniky molekulární MeSH
• exony MeSH
• introny MeSH
• klinické protokoly MeSH
• molekulární sekvence - údaje MeSH
• neuromuskulární nemoci diagnóza   genetika   MeSH
• sekvenční analýza DNA MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• neurologie
• ortopedie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Undercooked lamb and mutton are common sources of Toxoplasma gondii infection for humans. A sequence specific magnetic capture technique in combination with quantitative real-time PCR targeting the 529 bp repeat element of T. gondii was used for estimation of the parasite burdens in various sheep tissues (n = 6) three months after peroral experimental inoculation with 10,000 T. gondii oocysts. Brain was the most frequently affected organ (positive in all 6 sheep) and showed the highest estimated parasite loads (0.5-30,913 parasites/g tissue). Lung samples were positive in three sheep, with load estimates of 36.3 to <1 parasite/g tissue. Heart tissue was positive in three sheep and kidney only in one animal with low parasite loads (<1 parasite/g tissue). Only few skeletal muscle samples in 2 animals showed positive results, with very low parasite burdens, while samples from further internal organs (i.e. liver and spleen) were negative in all animals. This study identified the brain as the most important predilection site and therefore the most appropriate tissue for T. gondii detection.

• MeSH
• anatomické struktury zvířat parazitologie   MeSH
• kvantitativní polymerázová řetězová reakce přístrojové vybavení   metody   MeSH
• magnetismus MeSH
• ovce MeSH
• Toxoplasma genetika   izolace a purifikace   MeSH
• toxoplazmóza zvířat parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Undercooked meat containing tissue cysts is one of the most common sources of Toxoplasma gondii infection in humans. Goats are very susceptible to clinical toxoplasmosis, and especially kids are common food animals, thereby representing a risk for human infection. A sequence-specific magnetic capture method was used for isolation of T. gondii DNA from tissue samples from experimentally infected goat-kids and real-time PCR for the 529 bp repeat element allowed quantification of T. gondii DNA. The contamination level in different types of tissue and in two groups of goats euthanized 30 and 90 dpi was compared. The highest concentration of T. gondii DNA in both groups of goats was found in lung tissue, but only the higher parasite count in lung tissue compared to other organs in group A (euthanized 30 dpi) was statistically significant. T. gondii concentrations were higher in liver and dorsal muscle samples from goats euthanized 90 dpi than in goats euthanized at 30 dpi, while the T. gondii concentration in hearts decreased. This study describes for the first time distribution of T. gondii parasites in post-weaned goat kids. New information about T. gondii predilection sites in goats and about the progression of infection between 30 and 90 dpi was achieved.

• MeSH
• játra parazitologie   MeSH
• kosterní svaly parazitologie   MeSH
• kozy MeSH
• kvantitativní polymerázová řetězová reakce metody   veterinární   MeSH
• magnetismus MeSH
• mozek parazitologie   MeSH
• nemoci koz diagnóza   parazitologie   MeSH
• plíce parazitologie   MeSH
• slezina parazitologie   MeSH
• srdce parazitologie   MeSH
• Toxoplasma izolace a purifikace   MeSH
• toxoplazmóza zvířat diagnóza   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH