High-throughput DNA Sequencing Methods Research Articles

The Role of Bacteria in Pink Stone Discoloration: Insights from Batalha Monastery

The colonization of historical buildings and monuments by fungi, algae, and bacteria is a common phenomenon. This often leads to deterioration processes that cause either visual or structural harm. The Batalha Monastery in Portugal, a UNESCO World Heritage Site, currently shows significant surface changes to the stone architectural elements within both the Founder’s Chapel and the church, including a widespread pink discoloration on the walls and columns. The main goal of this study was to analyze the biological colonization and assess the influence of bacterial communities on the biodeterioration of Ançã limestone, providing valuable insights to help conservators and restorers select the best preservation strategies for the monastery. The prokaryote population was characterized using both high-throughput DNA sequencing and culture-dependent methods and several orange-pink pigment-producing bacteria were identified, for example, Bacillus, Gordonia, Serratia and Methylobacterium, as well as Halalkalicoccus, an abundant archaeal genus. The pink discoloration observed could be due to biofilms created by bacteria that produce pigments, namely carotenoids. Biocolonization tests were performed using stone mock-ups, which were prepared and inoculated with the bacteria isolated in this study. These tests were designed to replicate the natural conditions of the monastery and monitor the colonization process to understand the discoloration phenomenon.

A search for bacteria identified from cerebrospinal fluid shunt infections in previous surgical events.

Shunt infections are a common complication when treating hydrocephalus by cerebrospinal fluid (CSF) shunt placement. The source of infecting pathogens is not well understood. One hypothesis, which we explored here, is that microorganisms persist chronically in the host long before a symptomatic infection occurs and may be detectable in surgical events preceding infection. A cohort of 13 patients was selected, for which CSF samples were available from an infection episode and from a previous surgery event, which was either an initial shunt placement or a revision. Microbiota were analyzed both directly from CSF and from isolates cultured from CSF on aerobic and anaerobic media. The detection and identification of bacteria was done with high throughput DNA sequencing methods and mass spectrometry. The presence of bacteria was confirmed in 4 infection samples, of which 2 were after initial placement and 2 after revision surgery. Taxonomic identification was consistent with clinical microbiology laboratory results. Bacteria were not detected in any of the CSF samples collected at the time of the previous surgical events. While our findings do not provide direct evidence for long-term persistence of pathogens, they suggest the need for consideration of additional source material, such as biofilm and environmental swabs, and/or the use of more sensitive and specific analytical methods.

A high-throughput sequencing approach identifies immunotherapeutic targets for bacterial meningitis in neonates.

Worldwide, Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but full understanding of the pathogenesis of this disease is not yet achieved. Moreover, to date, no vaccine is available against bacterial neonatal meningitis. Here, we used Transposon Sequencing of saturated banks of mutants (TnSeq) to evaluate E.coli K1 genetic fitness in murine neonatal meningitis. We identified E.coli K1 genes encoding for factors important for systemic dissemination and brain infection, and focused on products with a likely outer-membrane or extra-cellular localization, as these are potential vaccine candidates. We used invitro and invivo models to study the efficacy of active and passive immunization. We selected for further study the conserved surface polysaccharide Poly-β-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E.coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and invitro these antibodies were also able to decrease binding, invasion and crossing of E.coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect invitro and invivo against this major neonatal pathogen. Altogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections. ANR Seq-N-Vaq, Charles Hood Foundation, Hearst Foundation, and Groupe Pasteur Mutualité.

Cyclobutane Pyrimidine Dimer Hyperhotspots as Sensitive Indicators of Keratinocyte UV Exposure†.

The dominant DNA damage generated by UV exposure is the cyclobutane pyrimidine dimer (CPD), which alters skin cell physiology and induces cell death and mutation. Genome-wide nucleotide-resolution analysis of CPDs in melanocytes and fibroblasts has identified "CPD hyperhotspots", pyrimidine-pyrimidine sites hundreds of fold more susceptible to the generation of CPDs than the genomic average. Identifying hyperhotspots in keratinocytes could enable measuring individual past UV exposure in small skin samples and predicting future skin cancer risk. We therefore exposed neonatal human epidermal keratinocytes to narrowband UVB and quantified CPDs using the adductSeq high-throughput DNA sequencing method. Keratinocytes contained thousands of CPD hyperhotspots, with a UVB-sensitivity up to 550 fold greater than the genomic average. As with melanocytes, the most sensitive sites were located in promoter regions at ETS-family transcription factor binding sequence motifs, near RNA processing genes. Moreover, they lay at sequence motifs bound to ETS1 in CpG islands. These genes were specifically upregulated in skin and the CPD hyperhotspots were mutated in a fraction of keratinocyte cancers. Crucially for their biological importance and practical application, CPD hyperhotspot locations and UV-sensitivity ranking demonstrated high reproducibility across experiments and across skin donors. CPD hyperhotspots are therefore sensitive indicators of UV exposure.

Ketoconazole 2% cream alters the skin fungal microbiome in seborrhoeic dermatitis: a cohort study.

Seborrhoeic dermatitis (SD) is a common chronic inflammatory dermatosis. Current theories on the pathogenesis of SD highlight the role of microbes on the skin surface. Ketoconazole is commonly used for the treatment of SD; however, there are limited data focusing on the effects of ketoconazole in shaping the skin microbiome in patients with SD. In this prospective cohort study, we used a high-throughput DNA sequencing method to characterize the cutaneous microbial communities of patients with SD before and after topical ketoconazole treatment. In total, 30 patients with facial SD and 15 age- and sex-matched healthy controls (HCs) were enrolled in this study. Skin swabs were collected from SD lesional sites of the cheek at baseline, after ketoconazole treatment and 2 weeks post-treatment. DNA was extracted from skin samples. The bacterial 16S V3V4 rRNA and fungal internal transcribed spacer 1-5F regions were sequenced, and the microbial community compositions were analysed. Significantly lower bacterial and fungal diversities were detected at the lesional sites of facial SD compared with HCs. A decreased relative abundance of Cutibacterium and increased abundances of Malassezia and Staphylococcus were found in facial SD. Disease diversity was positively correlated with the relative abundances of Malassezia, Staphylococcus and Corynebacterium, while transepidermal water loss was negatively associated with the relative abundance of Cutibacterium. After ketoconazole treatment, fungal Shannon diversity and the relative abundances of Candida and Aspergillus were significantly increased at the lesional sites, and the relative abundance of Malassezia showed a decreasing trend. These changing trends were maintained until 2 weeks post-treatment. Facial SD showed lower fungal diversity accompanied by increased relative abundances of Malassezia and Staphylococcus and decreased relative abundance of Cutibacterium. Ketoconazole treatment reduced Malassezia and increased fungal diversity to restore skin microbial communities.

A novel swab storage gel is superior to dry swab DNA collection, and enables long-range high resolution next generation sequencing HLA typing from buccal cell samples.

HLA sequence-based DNA typing (SBT) by long-range PCR amplification (LR PCR) and next-generation sequencing (NGS) is a high-throughput DNA sequencing method (LR-NGS-SBT) for the efficient and sensitive detection of novel and null HLA alleles to the field-4 level of allelic resolution without phase ambiguity. However, the accuracy and reliability of the HLA typing results using buccal cells (BCs) and saliva as genetic source materials for the LR-NGS-SBT method are dependent largely on the quality of the extracted genomic DNA (gDNA) because a large degree of gDNA fragmentation can result in insufficient PCR amplification with the incorrect assignment of HLA alleles because of allele dropouts. In this study, we developed a new cost-efficient swab storage gel (SSG) for wet swab collection of BCs (BC-SSG) and evaluated its usefulness by performing different DNA analytical parameters including LR-NGS-SBT to compare the quality and quantity of gDNA extracted from BCs (in SSG or air dried), blood and saliva of 30 subjects. The BC-SSG samples after 5 days of storage revealed qualitative and quantitative DNA values equivalent to that of blood and/or saliva and better than swabs that were only air-dried (BC-nSSG). Moreover, all the gDNA extracted from blood, saliva and BC-SSG samples were HLA-typed successfully to an equivalent total of 408 alleles for each sample type. Therefore, the application of BC-SSG collection media for LR-NGS-SBT has benefits over BC dried samples (dry swabs) such as reducing retesting and the number of untestable BC samples because of insufficient DNA amplification.

Update on CD164 and LMX1A genes to strengthen their causative role in autosomal dominant hearing loss.

Novel hearing loss (HL) genes are constantly being discovered, and evidence from independent studies is essential to strengthen their position as causes of hereditary HL. To address this issue, we searched our genetic data of families with autosomal dominant HL (ADHL) who had been tested with high-throughput DNA sequencing methods. For CD164, only one pathogenic variant in one family has so far been reported. For LMX1A, just two previous studies have revealed its involvement in ADHL. In this study we found two families with the same pathogenic variant in CD164 and one family with a novel variant in LMX1A (c.686C>A; p.(Ala229Asp)) that impairs its transcriptional activity. Our data show recurrence of the same CD164 variant in two HL families of different geographic origin, which strongly suggests it is a mutational hotspot. We also provide further evidence for haploinsufficiency as the pathogenic mechanism underlying LMX1A-related ADHL.

The Neglected Gut Microbiome: Fungi, Protozoa, and Bacteriophages in Inflammatory Bowel Disease.

The gut microbiome has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Studies suggest that the IBD gut microbiome is less diverse than that of the unaffected population, a phenomenon often referred to as dysbiosis. However, these studies have heavily focused on bacteria, while other intestinal microorganisms—fungi, protozoa, and bacteriophages—have been neglected. Of the nonbacterial microbes that have been studied in relation to IBD, most are thought to be pathogens, although there is evidence that some of these species may instead be harmless commensals. In this review, we discuss the nonbacterial gut microbiome of IBD, highlighting the current biases, limitations, and outstanding questions that can be addressed with high-throughput DNA sequencing methods. Further, we highlight the importance of studying nonbacterial microorganisms alongside bacteria for a comprehensive view of the whole IBD biome and to provide a more precise definition of dysbiosis in patients. With the rise in popularity of microbiome-altering therapies for the treatment of IBD, such as fecal microbiota transplantation, it is important that we address these knowledge gaps to ensure safe and effective treatment of patients.

Mobile mud layer underneath the desiccated maar lake of Rincón de Parangueo and insights into its microbial fingerprints

We surveyed a subsurface layer of saturated mobile mud and its microbial fingerprints below the desiccated bottom of a maar lake at Rincón de Parangueo (RP), Mexico. A multi-scale approach was followed using geological fieldwork, coring of the sediments, ground penetrating radar survey, physico-chemical characterization including X-ray diffraction and scanning electron microscope, and high-throughput DNA sequencing methods. The mobile mud is an organic-rich silty clay, with high values of alkalinity, volumetric water content and conductivity. Mud mobility has been attributed to overpressure caused by disequilibrium compaction of sediments related to active subsidence and pore overpressure produced by an input of groundwater and gas content in the sediments and resulted in a diverse set of structures related to mud tectonics such as injection domes and fluid seeps through fractures. Extraction and sequencing of sedimentary environmental DNA in the mud layer were performed for Bacteria and Archaea. Despite the small number of samples obtained, the microbial fingerprint from the sedimentary environmental DNA at subsurface shares similarities with the microbial communities identified on the crater surface. Additionally, we identify the DNA of specific methanogenic microorganisms in the mud, such as Bathyarchaeia, Methanomassiliicoccales, and Methanobacteriales, and we speculate on their probable role in gas production and pore overpressure in the mud layer. The underground mud at Rincón de Parangueo represents a geologically dynamic environment with conditions that are favorable for the thriving of microbial communities.

Reconstruction of evolving gene variants and fitness from short sequencing reads.

Directed evolution can generate proteins with tailor-made activities. However, full-length genotypes, their frequencies, and fitnesses are difficult to measure for evolving gene-length biomolecules using most high-throughput DNA sequencing methods as short read lengths can lose mutation linkages in haplotypes. We present Evoracle, a machine learning method that accurately reconstructs full-length genotypes (R2 = 0.94) and fitness using short-read data from directed evolution experiments, with substantial improvements over related methods. We validate Evoracle on phage-assisted continuous evolution (PACE), phage-assisted non-continuous evolution (PANCE) of adenine base editors, and OrthoRep evolution of drug-resistant enzymes. Evoracle retains strong performance (R2 = 0.86) on data with complete linkage loss between neighboring nucleotides and large measurement noise such as pooled Sanger sequencing data (~$10/timepoint), and broadens the accessibility of training machine learning models on gene variant fitnesses. Evoracle can also identify high-fitness variants, including low-frequency ‘rising stars’, well before they are identifiable from consensus mutations.

Next-Generation Sequencing Based Monitoring of Circulating-Tumor DNA in Untreated Peripheral T-Cell Lymphoma

Background: Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with a wide range of clinical outcomes. Overall prognosis is worse for PTCL than B-cell lymphomas, but pts who achieve remission can be cured. After initial therapy, controversy exists regarding consolidation with autologous stem cell transplant, and clinical decisions are largely empirical. Circulating-tumor DNA (ctDNA) is emerging as a promising non-invasive biomarker that may aid clinical decision-making. Quantitative baseline levels of ctDNA and kinetics of ctDNA clearance during therapy may provide prognostic information. Further, novel methods that detect disease relapse prior to imaging may improve outcomes. We studied a high-throughput DNA sequencing method of detecting T-cell receptor (TCR) β and γ gene sequences in serum with a sensitivity of 1 lymphoma molecule per million leukocytes. Using this platform, we determined the predictive ability of monitoring ctDNA in pts undergoing frontline therapy for PTCL.Methods: All pts had PTCL undergoing frontline EPOCH-based chemotherapy between May 1993 through December 2015. Pre-treatment FFPE biopsy samples and/or baseline serum samples were sent for VDJ amplification and TCR sequencing calibration. High-throughput TCR sequencing of the CDR3s of TCRβ/TCRγ genes was employed to provide a comprehensive and quantitative analysis of distinct T-cell clones within each sample, the relative frequency of each clone, and the exact unique nucleotide sequences of each clone's CDR3 regions. Pts with a calibrating TCRβ/TCRγ sequence had all available serum samples sent for identification and quantification. Prospectively collected serum sample time points included baseline, start of each cycle, and each follow-up visit until disease progression. Clinic visits after therapy included paired contrast CT scans performed at 3, 4, 6, 12 and 12 mos. intervals for the first 5 years, respectively. All analyses for ctDNA were performed blinded to clinical outcomes.Results: Fifty PTCL pts had a pre-treatment FFPE or serum sample available for calibration. Nineteen of 23 (83%) pts with pre-treatment FFPE had a calibrating rearrangement; Fourteen of 27 (52%) pts with only pre-treatment serum available had a calibrating rearrangement; 1 pt who did not calibrate on FFPE had a dominant sequence identified in serum. Overall, tumor-specific clonotypes were identified in 34 (68%) of 50 pts tested. Ten of 18 (56%) pts with both FFPE and serum available had dominant clones in both compartments. Of the 44 calibrating pre-treatment samples, 1 calibrated for TCRβ only, 15 for TCRγ only, and 28 for both loci. Pt characteristics shown in Table 1. Of 34 study pts, 20 (59%) progressed and 14 (41%) were non-progressors. Quantitative baseline ctDNA levels were significantly higher in pts with stage IV disease compared to stages I-III (p=0.014) but did not correlate with LDH levels (p=0.53).We analyzed the predictive ability of ctDNA as a measure of minimal residual disease (MRD) within 1 month of completing therapy in 28 pts. We compared pts who became MRD- to those who remained MRD+ and identified a trend towards improved PFS in those who became MRD- (median, NR vs. 208 days, p=0.07) (Figure 1). In 20 pts who achieved remission, we studied the use of ctDNA monitoring as a surveillance tool. Of the 10 pts who progressed after remission, ctDNA was detected prior to clinical relapse in 6 cases with a median lead-time of 13.2 mos. (range, 2.7 to 88.5) and 4 with detectable ctDNA > 12 mos. prior to clinical relapse. Four pts progressed without detectable ctDNA prior to clinical relapse. Interestingly, of the 10 non-progressors followed in surveillance, 5 pts developed low-level ctDNA positivity without clinical progression. Results of interim ctDNA kinetics and TCRβ testing is ongoing and will be presented at the meeting.Conclusions: Next-generation based sequencing of the TCR can identify tumor-specific clonotypic sequences in the majority of pts from both FFPE and serum. As a marker of MRD, monitoring TCR sequences in serum may identify pts who do not benefit from treatment intensification, but further studies are needed. Surveillance monitoring of ctDNA in serum identifies TCR sequences months before clinical progression but not all pts with clonotypic sequences progressed clinically in our series. Further studies are needed to investigate surveillance monitoring methods and optimize clinical utility. [Display omitted] DisclosuresJacob:Adaptive Biotechnologies: Employment, Equity Ownership. Chan:Adaptive Biotechnologies: Employment. Vignali:Adaptive Biotechnologies: Employment.

There's always a better way: The application of eDNA to effectively assess biodiversity

Our ecosystem monitoring methodologies focus on data collection for reporting purposes that may not serve to identify the systematic causes of ecological change. Managers need precise and timely information at appropriate scales to build ecosystem resilience. Traditional species detection methodologies offer little information when species abundance are low, especially in large water ecosystems such as the Great Lakes. Species not found during monitoring doesn’t necessarily mean that species are absent. Moreover, even if a change in the ecosystem is detected, it is often not possible to determine its cause at a spatiotemporal scale or a trophic cascade level. As a result, we often find ourselves being reactive in our mitigation measures. Before irreversible change occurs, we must be guided by a better understanding of the actual ecological landscape which Environmental DNA (eDNA) may help provide. eDNA is a potential tool to effectively overcome traditional species survey limitations currently in use at many Parks Canada sites (Supplemental file 1). As various organisms interact with the environment, DNA is expelled and accumulates in their surroundings. Such samples can be analyzed by high-throughput DNA sequencing methods for rapid measurement and monitoring of biodiversity. Access to this genetic information makes a critical contribution to the understanding of population size, species distribution, and population dynamics for species not well documented. Despite the increasing use of eDNA in conservation practice, it requires further methodological improvement for greater influence on management decisions. The tool requires standardized protocols based on site-specific covariates and objectives. We’re working to tackle the challenge with 2 objectives: (1) to combine traditional biomonitoring knowledge and metagenomics to further develop eDNA as a reliable sampling tool for Parks Canada and (2) to support site-specific monitoring objectives for species-at-risk, invasive species, aquatic species inventories, and/or culturally significant species. The overall goal is to increase our capacity to make more informed, timely, regionally-coordinated conservation decisions through the rapid and sensitive species detection methods offered by eDNA.

Shifts of microbiota during cheese production: impact on production and quality.

The high-throughput DNA sequencing (HTS) method is used to identify microbes in cheese and their potential functional properties. The technique can be applied to the microbiota of the cheese processing environment, raw milk, curd, whey, and starter cultures, and be used to improve the quality, safety, and other physicochemical properties of the final product. The HTS method is also utilized to study the microbiota shift of different types of cheeses during processing, as the composition and functional properties of the microbiome provide unique characteristics to different cheeses. Although there are several reviews that focused on microbiota of various types of cheeses, this review focuses on evaluating the microbiota shift of different types of cheese production and highlights key bacteria in each step of the processing as well as microbiota of various types of cheeses. KEY POINTS: • High-throughput sequencing can be applied to identify microbiota in cheese. • Microbiota in cheese is changed during making process and aging. • Starter culture plays an important role to establish microbiota in cheese.

An Overview of Bioinformatics Tools for DNA Meta-Barcoding Analysis of Microbial Communities of Bioaerosols: Digest for Microbiologists.

High-throughput DNA sequencing (HTS) has changed our understanding of the microbial composition present in a wide range of environments. Applying HTS methods to air samples from different environments allows the identification and quantification (relative abundance) of the microorganisms present and gives a better understanding of human exposure to indoor and outdoor bioaerosols. To make full use of the avalanche of information made available by these sequences, repeated measurements must be taken, community composition described, error estimates made, correlations of microbiota with covariates (variables) must be examined, and increasingly sophisticated statistical tests must be conducted, all by using bioinformatics tools. Knowing which analysis to conduct and which tools to apply remains confusing for bioaerosol scientists, as a litany of tools and data resources are now available for characterizing microbial communities. The goal of this review paper is to offer a guided tour through the bioinformatics tools that are useful in studying the microbial ecology of bioaerosols. This work explains microbial ecology features like alpha and beta diversity, multivariate analyses, differential abundances, taxonomic analyses, visualization tools and statistical tests using bioinformatics tools for bioaerosol scientists new to the field. It illustrates and promotes the use of selected bioinformatic tools in the study of bioaerosols and serves as a good source for learning the “dos and don’ts” involved in conducting a precise microbial ecology study.

High-throughput sequencing of IgG B-cell receptors reveals frequent usage of the rearranged IGHV4\u201328/IGHJ4 gene in primary immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4–28 in ITP patients. One hundred eighty-six distinct IGHV4–28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4–28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4–28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4–28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.

Advancements in MAPK signaling pathways and MAPK-targeted therapies for ameloblastoma: A review.

Numerous signal transduction pathways are closely associated with the occurrence, development, and prognosis of ameloblastoma (AM). Mitogen-activated protein kinase (MAPK) is a serine/threonine-specific protein kinase that transduces intracellular signals in critical cellular phenomena. A number of recent analyses have reported that the MAPK signaling pathway contributes significantly to AM. High-throughput DNA sequencing methods, such as next-generation sequencing using Illumina have yielded advancements in studies on MAPK signaling pathways and their association with AM; in particular, BRAF V600E is mediated by the activation of the Ras/Raf/MAPK pathway. This review discusses advancements in studies on MAPK signaling pathways and MAPK-targeted inhibitors or antibodies, along with the merits and demerits of MAPK-targeted therapies, finally followed by a discussion regarding more efficient potential MAPK-targeted therapies to treat AM with few side effects, thereby providing novel insights into targeted therapy for AM.

Uncovering the hidden microbiota in hospital and built environments: New approaches and solutions.

Research concerning the microbiome of indoor environments like hospitals, houses or buildings could have several implications for human health. Today, there is an ongoing shift in the paradigm of microbial analysis, from single isolated bacterial samples to entire microbiome profiles using high-throughput DNA sequencing methods. The use of sequencing methods in several studies has revealed an unprecedented microbial diversity in indoor environments, leading to a larger comprehension of the entire microbiome context. Here, we present a review of these microbiome studies using high-throughput DNA sequencing, including some new approaches and ideas that can be broadly applied in microbial tracking and epidemiological surveillance of indoor environments.

Zastosowanie technologii wysokoprzepustowego sekwencjonowania DNA w genetyce sądowej

The turn of the 20th and 21st centuries marks the beginning of high-throughput DNA sequencing methods, which, owing to increasing efficiency and gradual cost reduction, have led to the revolutionization of biomedical research. This article discusses the most popular next generation sequencing technologies and their practical application in forensic genetic analysis.

Gene expression analysis and in vitro production procedures for bovine preimplantation embryos: Past highlights, present concepts and future prospects.

Over the past decades in vitro production (IVP) of bovine embryos has been significantly improved and in particular bovine IVP is now widely applied under field conditions. This in vitro technique provides new opportunities for cattle producers, particularly in the dairy industry, to overcome infertility and to increase dissemination of animals with high genetic merit. Improvements in OPU/IVP resulted in large-scale international commercialization. More than half a million IVP embryos are generated on the yearly basis demonstrating the enormous potential of this technology. These advances and the fact that bovine and human early development is remarkably similar have also prompted the use of bovine embryos as a model system to study early mammalian embryogenesis including humans. Despite all the improvements, embryos generated in vitro still differ from their in vivo derived counterparts. Embryos must adjust to multiple microenvironments at preimplantation stages. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting embryo. The successful clinical application of the techniques in reproductive biotechnology requires both species-specific clinical skills and extensive laboratory experience. The recent advances in transcriptome profiling have also provided deeper insight into RNA expression and regulation at an unprecedented resolution. The development of these high-throughput DNA sequencing methods has resulted in new approaches for both mapping and quantifying transcriptomes. The aim of this review is therefore to summarize the available data related to gene expression analyses as well as in vitro embryo production procedures and to provide ideas about future concepts.

Bifidobacterium pseudolongum in the Ceca of Rats Fed Hi-Maize Starch Has Characteristics of a Keystone Species in Bifidobacterial Blooms.

Starches resistant to mammalian digestion are present in foods and pass to the large bowel, where they may be degraded and fermented by the microbiota. Increases in relative abundances of bifidobacteria (blooms) have been reported in rats whose diet was supplemented with Hi-Maize resistant starch. We determined that the bifidobacterial species present in the rat cecum under these circumstances mostly belonged to Bifidobacterium animalis However, cultures of B. animalis isolated from the rats failed to degrade Hi-Maize starch to any extent. In contrast, Bifidobacterium pseudolongum also detected in the rat microbiota had high starch-degrading ability. Transcriptional comparisons showed increased expression of a type 1 pullulanase, alpha-amylase, and glycogen debranching enzyme by B. pseudolongum when cultured in medium containing Hi-Maize starch. Maltose was released into the culture medium, and B. animalis cultures had shorter doubling times in maltose medium than did B. pseudolongum Thus, B. pseudolongum, which was present at a consistently low abundance in the microbiota, but which has extensive enzymatic capacity to degrade resistant starch, showed the attributes of a keystone species associated with the bifidobacterial bloom.IMPORTANCE This study addresses the microbiology and function of a natural ecosystem (the rat gut) using DNA-based observations and in vitro experimentation. The microbial community of the large bowel of animals, including humans, has been studied extensively through the use of high-throughput DNA sequencing methods and advanced bioinformatics analysis. These studies reveal the compositions and genetic capacities of microbiotas but not the intricacies of how microbial communities function. Our work, combining DNA sequence analysis and laboratory experiments with cultured strains of bacteria, revealed that the increased abundance of bifidobacteria in the rat gut, induced by feeding indigestible starch, involved a species that cannot itself degrade the starch (Bifidobacterium animalis) but cohabits with a species that can (Bifidobacterium pseudolongum). B. pseudolongum has the characteristics of a keystone species in the community because it had low abundance but high ability to perform a critical function, the hydrolysis of resistant starch.