576 Genetik und Evolution
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Trade of wild-caught animals is illegal for many taxa and in many countries. Common regulatory procedures involve documentation and marking techniques. However, these procedures are subject to fraud and thus should be complemented by routine genetic testing in order to authenticate the captive-bred origin of animals intended for trade. A suitable class of genetic markers are SNPSTRs that combine a short tandem repeat (STR) and single nucleotide polymorphisms (SNPs) within one amplicon. This combined marker type can be used for genetic identification and for parentage analyses and in addition, provides insight into haplotype history. As a proof of principle, this study establishes a set of 20 SNPSTR markers for Athene noctua, one of the most trafficked owls in CITES Appendix II. These markers can be coamplified in a single multiplex reaction. Based on population data, the percentage of observed and expected heterozygosities of the markers ranged from 0.400 to 1.000 and 0.545 to 0.850, respectively. A combined probability of identity of 5.3*10-23 was achieved with the whole set, and combined parentage exclusion probabilities reached over 99.99%, even if the genotype of one parent was missing. A direct comparison of an owl family and an unrelated owl demonstrated the applicability of the SNPSTR set in parentage testing. The established SNPSTR set thus proved to be highly useful for identifying individuals and analysing parentage to determine wild or captive origin. We propose to implement SNPSTR-based routine certification in wildlife trade as a way to reveal animal laundering and misdeclaration of wild-caught animals.
Background: Bloodstream infections (BSIs) remain a significant cause of mortality worldwide. Causative pathogens are routinely identified and susceptibility tested but only very rarely investigated for their resistance genes, virulence factors, and clonality. Our aim was to gain insight into the clonality patterns of different species causing BSI and the clinical relevance of distinct virulence genes.
Methods: For this study, we whole-genome-sequenced over 400 randomly selected important pathogens isolated from blood cultures in our diagnostic department between 2016 and 2021. Genomic data on virulence factors, resistance genes, and clonality were cross-linked with in-vitro data and demographic and clinical information.
Results: The investigation yielded extensive and informative data on the distribution of genes implicated in BSI as well as on the clonality of isolates across various species.
Conclusion: Associations between survival outcomes and the presence of specific genes must be interpreted with caution, and conducting replication studies with larger sample sizes for each species appears mandatory. Likewise, a deeper knowledge of virulence and host factors will aid in the interpretation of results and might lead to more targeted therapeutic and preventive measures. Monitoring transmission dynamics more efficiently holds promise to serve as a valuable tool in preventing in particular BSI caused by nosocomial pathogens.
Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance.
The development of whole-genome amplification (WGA) techniques has opened up new avenues for genetic analysis and genome research, in particular by facilitating the genome-wide analysis of few or even single copies of genomic DNA, such as from single cells (prokaryotic or eukaryotic) or virions. Using WGA, the few copies of genomic DNA obtained from such entities are unspecifically amplified using PCR or PCR-related processes in order to obtain higher DNA quantities that can then be successfully analysed further.
Cyanobacteria are gaining considerable interest as a method of supporting the long-term presence of humans on the Moon and settlements on Mars due to their ability to produce oxygen and their potential as bio-factories for space biotechnology/synthetic biology and other applications. Since many unknowns remain in our knowledge to bridge the gap and move cyanobacterial bioprocesses from Earth to space, we investigated cell division resumption on the rehydration of dried Chroococcidiopsis sp. CCMEE 029 accumulated DNA damage while exposed to space vacuum, Mars-like conditions, and Fe-ion radiation. Upon rehydration, the monitoring of the ftsZ gene showed that cell division was arrested until DNA damage was repaired, which took 48 h under laboratory conditions. During the recovery, a progressive DNA repair lasting 48 h of rehydration was revealed by PCR-stop assay. This was followed by overexpression of the ftsZ gene, ranging from 7.5- to 9-fold compared to the non-hydrated samples. Knowing the time required for DNA repair and cell division resumption is mandatory for deep-space experiments that are designed to unravel the effects of reduced/microgravity on this process. It is also necessary to meet mission requirements for dried-sample implementation and real-time monitoring upon recovery. Future experiments as part of the lunar exploration mission Artemis and the lunar gateway station will undoubtedly help to move cyanobacterial bioprocesses beyond low Earth orbit. From an astrobiological perspective, these experiments will further our understanding of microbial responses to deep-space conditions.
Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.
When the Artemis missions launch, NASA's Orion spacecraft (and crew as of the Artemis II mission) will be exposed to the deep space radiation environment beyond the protection of Earth's magnetosphere. Hence, it is essential to characterize the effects of space radiation, microgravity, and the combination thereof on cells and organisms, i.e., to quantify any correlations between the deep space radiation environment, genetic variation, and induced genetic changes in cells. To address this, the Artemis I mission will include the Peristaltic Laboratory for Automated Science with Multigenerations (PLASM) hardware containing the Deep Space Radiation Genomics (DSRG) experiment. The scientific aims of DSRG are (i) to identify the metabolic and genomic pathways in yeast affected by microgravity, space radiation, and their combination, and (ii) to differentiate between gravity and radiation exposure on single-gene deletion/overexpressing strains' ability to thrive in the spaceflight environment. Yeast is used as a model system because 70% of its essential genes have a human homolog, and over half of these homologs can functionally replace their human counterpart. As part of the experiment preparation towards spaceflight, an Experiment Verification Test (EVT) was performed at the Kennedy Space Center to verify that the experiment design, hardware, and approach to automated operations will enable achieving the scientific aims. For the EVT, fluidic systems were assembled, sterilized, loaded, and acceptance-tested, and subsequently integrated with the engineering parts to produce a flight-like PLASM unit. Each fluidic system consisted of (i) a Media Bag, (ii) four Culture Bags loaded with Saccharomyces cerevisiae (two with deletion series and the remaining two with overexpression series), and (iii) tubing and check valves. The EVT PLASM unit was put under a temperature profile replicating the anticipated different phases of flight, including handover to launch, spaceflight, and splashdown to handover back to the science team, for a 58-day period. At EVT completion, the rate of activation, cellular growth, RNA integrity, and sample contamination were interrogated. All of the experiment's success criteria were satisfied, encouraging our efforts to perform this investigation on Artemis I. This manuscript thus describes the process of spaceflight experiment design maturation with a focus on the EVT, its results, DSRG's preparation for its planned launch on Artemis I in 2022, and how the PLASM hardware can enable other scientific goals on future Artemis missions and/or the Lunar Orbital Platform – Gateway.
Modern PCR-based analytical techniques have reached sensitivity levels that allow for obtaining complete forensic DNA profiles from even tiny traces containing genomic DNA amounts as small as 125 pg. Yet these techniques have reached their limits when it comes to the analysis of traces such as fingerprints or single cells. One suggestion to overcome these limits has been the usage of whole genome amplification (WGA) methods. These methods aim at increasing the copy number of genomic DNA and by this means generate more template DNA for subsequent analyses. Their application in forensic contexts has so far remained mostly an academic exercise, and results have not shown significant improvements and even have raised additional analytical problems. Until very recently, based on these disappointments, the forensic application of WGA seems to have largely been abandoned. In the meantime, however, novel improved methods are pointing towards a perspective for WGA in specific forensic applications. This review article tries to summarize current knowledge about WGA in forensics and suggests the forensic analysis of single-donor bioparticles and of single cells as promising applications.