Development of methods for the characterization and retrieval of damaged DNA from ancient biological material

Development of methods for the characterization and retrieval of damaged DNA from ancient biological material Over course of the last decade, the field of ancient DNA has been transformed by the advent of highthroughput sequencing. In specimens with exceptional DNA preservation, this allowed whole nuclear genomes of extinct organisms to be sequenced and analyzed. To deal with material with suboptimal DNA preservation and high levels of external modern DNA contamination as is common in intensively handled museum specimens, significant challenges remain. In this thesis I developed laboratory and in silico methods to make highly contaminated biological material amenable to genetic research and explore the structure and decay mechanisms of ancient DNA. Chapter 1 illustrates the development of a library preparation method for high-throughput sequencing that selects for DNA molecules containing uracils, a damaged base typically found in ancent DNA, in order to enrich authentic ancient molecules against a background of modern contamination. The method was applied to Neanderthal samples from Gibraltar demonstrating its suitability for recovering DNA sequences from material that shows very poor DNA preservation and that is particularly strongly contaminated with modern human DNA. Chapter 2 describes a new method to reconstruct the native double-stranded DNA molecules and overhang structures in DNA isolated from a Neanderthal specimen by combining deep sequencing of low-input single-stranded DNA libraries with in silico sequence matching. I analyzed patterns of nucleotide substitutions and base composition separately in the different structures found and showed also that the method can be applied to modern sources of fragmented DNA, specifically to cell-free DNA isolated from present-day blood samples

Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual

A direct RT-qPCR approach to test large numbers of individuals for SARS-CoV-2

SARS-CoV-2 causes substantial morbidity and mortality in elderly and immunocompromised individuals, particularly in retirement homes, where transmission from asymptomatic staff and visitors may introduce the infection. Here we present a cheap and fast approach to detect SARSCoV-2 in single or pooled gargle lavages (“mouthwashes”). With this approach, we test all staff at a nursing home daily over a period of three weeks in order to reduce the risk that the infection penetrates the facility. This or similar approaches could be implemented to protect hospitals, nursing homes and other institutions in this and future viral epidemics

Development of methods for the characterization and retrieval of damaged DNA from ancient biological material

Development of methods for the characterization and retrieval of damaged DNA from ancient biological material Over course of the last decade, the field of ancient DNA has been transformed by the advent of highthroughput sequencing. In specimens with exceptional DNA preservation, this allowed whole nuclear genomes of extinct organisms to be sequenced and analyzed. To deal with material with suboptimal DNA preservation and high levels of external modern DNA contamination as is common in intensively handled museum specimens, significant challenges remain. In this thesis I developed laboratory and in silico methods to make highly contaminated biological material amenable to genetic research and explore the structure and decay mechanisms of ancient DNA. Chapter 1 illustrates the development of a library preparation method for high-throughput sequencing that selects for DNA molecules containing uracils, a damaged base typically found in ancent DNA, in order to enrich authentic ancient molecules against a background of modern contamination. The method was applied to Neanderthal samples from Gibraltar demonstrating its suitability for recovering DNA sequences from material that shows very poor DNA preservation and that is particularly strongly contaminated with modern human DNA. Chapter 2 describes a new method to reconstruct the native double-stranded DNA molecules and overhang structures in DNA isolated from a Neanderthal specimen by combining deep sequencing of low-input single-stranded DNA libraries with in silico sequence matching. I analyzed patterns of nucleotide substitutions and base composition separately in the different structures found and showed also that the method can be applied to modern sources of fragmented DNA, specifically to cell-free DNA isolated from present-day blood samples

Development of methods for the characterization and retrieval of damaged DNA from ancient biological material

Development of methods for the characterization and retrieval of damaged DNA from ancient biological material Over course of the last decade, the field of ancient DNA has been transformed by the advent of highthroughput sequencing. In specimens with exceptional DNA preservation, this allowed whole nuclear genomes of extinct organisms to be sequenced and analyzed. To deal with material with suboptimal DNA preservation and high levels of external modern DNA contamination as is common in intensively handled museum specimens, significant challenges remain. In this thesis I developed laboratory and in silico methods to make highly contaminated biological material amenable to genetic research and explore the structure and decay mechanisms of ancient DNA. Chapter 1 illustrates the development of a library preparation method for high-throughput sequencing that selects for DNA molecules containing uracils, a damaged base typically found in ancent DNA, in order to enrich authentic ancient molecules against a background of modern contamination. The method was applied to Neanderthal samples from Gibraltar demonstrating its suitability for recovering DNA sequences from material that shows very poor DNA preservation and that is particularly strongly contaminated with modern human DNA. Chapter 2 describes a new method to reconstruct the native double-stranded DNA molecules and overhang structures in DNA isolated from a Neanderthal specimen by combining deep sequencing of low-input single-stranded DNA libraries with in silico sequence matching. I analyzed patterns of nucleotide substitutions and base composition separately in the different structures found and showed also that the method can be applied to modern sources of fragmented DNA, specifically to cell-free DNA isolated from present-day blood samples

Toxins from scratch? Diverse, multimodal gene origins in the predatory robber fly Dasypogon diadema indicate a dynamic venom evolution in dipteran insects

BACKGROUND: Venoms and the toxins they contain represent molecular adaptations that have evolved on numerous occasions throughout the animal kingdom. However, the processes that shape venom protein evolution are poorly understood because of the scarcity of whole-genome data available for comparative analyses of venomous species. RESULTS: We performed a broad comparative toxicogenomic analysis to gain insight into the genomic mechanisms of venom evolution in robber flies (Asilidae). We first sequenced a high-quality draft genome of the hymenopteran hunting robber fly Dasypogon diadema, analysed its venom by a combined proteotranscriptomic approach, and compared our results with recently described robber fly venoms to assess the general composition and major components of asilid venom. We then applied a comparative genomics approach, based on 1 additional asilid genome, 10 high-quality dipteran genomes, and 2 lepidopteran outgroup genomes, to reveal the evolutionary mechanisms and origins of identified venom proteins in robber flies. CONCLUSIONS: While homologues were identified for 15 of 30 predominant venom protein in the non-asilid genomes, the remaining 15 highly expressed venom proteins appear to be unique to robber flies. Our results reveal that the venom of D. diadema likely evolves in a multimodal fashion comprising (i) neofunctionalization after gene duplication, (ii) expression-dependent co-option of proteins, and (iii) asilid lineage-specific orphan genes with enigmatic origin. The role of such orphan genes is currently being disputed in evolutionary genomics but has not been discussed in the context of toxin evolution. Our results display an unexpected dynamic venom evolution in asilid insects, which contrasts the findings of the only other insect toxicogenomic evolutionary analysis, in parasitoid wasps (Hymenoptera), where toxin evolution is dominated by single gene co-option. These findings underpin the significance of further genomic studies to cover more neglected lineages of venomous taxa and to understand the importance of orphan genes as possible drivers for venom evolution

Effect of iterative reconstruction and temporal averaging on contour sharpness in dynamic myocardial CT perfusion: Sub-analysis of the prospective 4D CT perfusion pilot study.

PURPOSE:Myocardial computed tomography perfusion (CTP) allows the assessment of the functional relevance of coronary artery stenosis. This study investigates to what extent the contour sharpness of sequences acquired by dynamic myocardial CTP is influenced by the following noise reduction methods: temporal averaging and adaptive iterative dose reduction 3D (AIDR 3D). MATERIALS AND METHODS:Dynamic myocardial CT perfusion was conducted in 29 patients at a dose level of 9.5±2.0 mSv and was reconstructed with both filtered back projection (FBP) and strong levels of AIDR 3D. Temporal averaging to reduce noise was performed as a post-processing step by combining two, three, four, six and eight original consecutive 3D datasets. We evaluated the contour sharpness at four distinct edges of the left-ventricular myocardium based on two different approaches: the distance between 25% and 75% of the maximal grey value (d) and the slope in the contour (m). RESULTS:Iterative reconstruction reduced contour sharpness: both measures of contour sharpness performed better for FBP than for AIDR 3D (d = 1.7±0.4 mm versus 2.0±0.5 mm, p>0.059 at all edges; m = 255.9±123.9 HU/mm versus 160.6±123.5 HU/mm; p0.052 at all edges and for m p<0.001 at all edges). CONCLUSION:The use of both temporal averaging and iterative reconstruction degrades objective contour sharpness parameters of dynamic myocardial CTP. Thus, further advances in image processing are needed to optimise contour sharpness of 4D myocardial CTP