Development and Characterisation of Passive CMOS Sensors for Pixel Detectors in High Radiation Environments

With the upgrade of the Large Hadron Collider (LHC) to the High-Luminosity LHC (HL-LHC), the instantaneous luminosity increases by a factor of five with respect to its design value from 2027. To keep the detectors at the HL-LHC operational in this harsh radiation environment with unprecedented hit rates and radiation levels, they undergo major upgrades. The ATLAS experiment replaces its current tracking detector by a large-area and all-silicon tracking detector consisting of silicon strip and hybrid pixel detectors (ATLAS ITk detector). This challenging upgrade requires the development of radiation tolerant technologies for both the readout electronics and the sensing part of the detector. In this thesis, the utilisation of a commercial CMOS process line for the production of sensors for hybrid pixel detectors is investigated. CMOS process lines offer high throughput at comparatively low costs and are thus a cost-effective solution for the production of large-area detectors. Further benefits arise from the availability of several features in the used CMOS technology like many metal layers, poly-silicon layers and MIM-capacitors which can help to enhance the sensor design. These features are mostly not available in conventional sensor productions. After an intensive R&D-programme over several years, large-area passive CMOS sensors compatible with the future readout chip of the ATLAS ITk pixel detector have been successfully manufactured and characterised for the first time. Within the scope of this work, the radiation tolerance (using protons) of passive CMOS sensors up to a fluence of 1e16 neq/cm2 is demonstrated. Furthermore, the performance of passive CMOS sensors before and after irradiation is studied in detail and compared with the sensor requirements for the future ATLAS ITk pixel detector. Crucial parameters like production yield, breakdown behaviour, electronic noise, hit-detection efficiency and charge collection behaviour are investigated. Passive CMOS sensors are found to meet the requirements for the ATLAS ITk pixel detector. In particular, an (in-time) hit-detection efficiency larger than 99% is measured after irradiation demonstrating the suitability of passive CMOS sensors for harsh radiation environments

Forensic SNP genotyping using nanopore MinION sequencing

One of the latest developments in next generation sequencing is the Oxford Nanopore Technologies' (ONT) MinION nanopore sequencer. We studied the applicability of this system to perform forensic genotyping of the forensic female DNA standard 9947 A using the 52 SNP-plex assay developed by the SNPforID consortium. All but one of the loci were correctly genotyped. Several SNP loci were identified as problematic for correct and robust genotyping using nanopore sequencing. All these loci contained homopolymers in the sequence flanking the forensic SNP and most of them were already reported as problematic in studies using other sequencing technologies. When these problematic loci are avoided, correct forensic genotyping using nanopore sequencing is technically feasible

Massively parallel sequencing of micro-manipulated cells targeting a comprehensive panel of disease-causing genes : a comparative evaluation of upstream whole-genome amplification methods

Single Gene Disorders (SGD) are still routinely diagnosed using PCR-based assays that need to be developed and validated for each individual disease-specific gene fragment. The TruSight One sequencing panel currently covers 12 Mb of genomic content, including 4813 genes associated with a clinical phenotype. When only a limited number of cells are available, whole genome amplification (WGA) is required prior to DNA target capture techniques such as the TruSight One panel. In this study, we compared 4 different WGA methods in combination with the TruSight One sequencing panel to perform single nucleotide polymorphism (SNP) genotyping starting from 3 micro-manipulated cells. This setting simulates clinical settings such as day-5 blastocyst biopsy for Preimplantation Genetic Testing (PGT), liquid biopsy of circulating tumor cells (CTCs) and cancer-cell profiling. Bulk cell samples were processed alongside these WGA samples to serve as a performance reference. Target coverage, coverage uniformity and SNP calling accuracy obtained using any of the WGA, is inferior to the results obtained on bulk cell samples. However, results after REPLI-g come close. Compared to the other WGA methods, the method using REPLI-g WGA results in a better coverage of the targeted genomic regions with a more uniform read depth. Consequently, this method also results in a more accurate SNP calling and could be considered for clinical genotyping of a limited number of cells

Multiplex STR amplification sensitivity in a silicon microchip

The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (mu PCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 mu l each. Diluted reference DNA samples (1 ng-31 pg) were amplified on the mu PCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both mu PCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis

Forensic massively parallel sequencing data analysis tool: implementation of MyFLq as a standalone web- and Illumina BaseSpace®-application

Routine use of massively parallel sequencing (MPS) for forensic genomics is on the horizon. The last few years, several algorithms and workflows have been developed to analyze forensic MPS data. However, none have yet been tailored to the needs of the forensic analyst who does not possess an extensive bioinformatics background. We developed our previously published forensic MPS data analysis framework MyFLq (My-Forensic-Loci-queries) into an open-source, user-friendly, web-based application. It can be installed as a standalone web application, or run directly from the Illumina BaseSpace environment. In the former, laboratories can keep their data on-site, while in the latter, data from forensic samples that are sequenced on an Illumina sequencer can be uploaded to Basespace during acquisition, and can subsequently be analyzed using the published MyFLq BaseSpace application. Additional features were implemented such as an interactive graphical report of the results, an interactive threshold selection bar, and an allele length-based analysis in addition to the sequenced-based analysis. Practical use of the application is demonstrated through the analysis of four 16-plex short tandem repeat (STR) samples, showing the complementarity between the sequence- and length-based analysis of the same MPS data

Nanopore sequencing of a forensic STR multiplex reveals loci suitable for single-contributor STR profiling

Nanopore sequencing for forensic short tandem repeats (STR) genotyping comes with the advantages associated with massively parallel sequencing (MPS) without the need for a high up-front device cost, but genotyping is inaccurate, partially due to the occurrence of homopolymers in STR loci. The goal of this study was to apply the latest progress in nanopore sequencing by Oxford Nanopore Technologies in the field of STR genotyping. The experiments were performed using the state of the art R9.4 flow cell and the most recent R10 flow cell, which was specifically designed to improve consensus accuracy of homopolymers. Two single-contributor samples and one mixture sample were genotyped using Illumina sequencing, Nanopore R9.4 sequencing, and Nanopore R10 sequencing. The accuracy of genotyping was comparable for both types of flow cells, although the R10 flow cell provided improved data quality for loci characterized by the presence of homopolymers. We identify locus-dependent characteristics hindering accurate STR genotyping, providing insights for the design of a panel of STR loci suited for nanopore sequencing. Repeat number, the number of different reference alleles for the locus, repeat pattern complexity, flanking region complexity, and the presence of homopolymers are identified as unfavorable locus characteristics. For single-contributor samples and for a limited set of the commonly used STR loci, nanopore sequencing could be applied. However, the technology is not mature enough yet for implementation in routine forensic workflows

Maternal recognition of pregnancy in the horse : are MicroRNAs the secret messengers?

The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo-maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro-RNA-Sequencing was performed on all collected samples, mRNA-Sequencing on the same tissue samples and mass spectrometry was conducted previously on the same fluid samples. Differential expression of miRNA, mRNA and proteins showed high conformity with literature and confirmed involvement in pregnancy establishment, embryo quality, steroid synthesis and prostaglandin regulation, but the link between differential miRNAs and their targets was limited and did not indicate the identity of an unequivocal signal for MRP in the horse. Differential expression at the embryo-maternal interface was prominent, highlighting a potential role of miRNAs in embryo-maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies

Forensic tri-allelic SNP genotyping using nanopore sequencing

The potential and current state-of-the-art of forensic SNP genotyping using nanopore sequencing was investigated with a panel of 16 tri-allelic single nucleotide polymorphisms (SNPs), multiplexing five samples per sequencing run. The sample set consisted of three single-source human genomic reference control DNA samples and two GEDNAP samples, simulating casework samples. The primers for the multiplex SNP-loci PCR were taken from a study which researched their value in a forensic setting using conventional single-base extension technology. Workflows for multiplexed Oxford Nanopore Technologies 1D and 1D(2) sequencing were developed that provide correct genotyping of most SNP loci. Loci that are problematic for nanopore sequencing were characterized. When such loci are avoided, nanopore sequencing of forensic tri-allelic SNPs is technically feasible

BDAQ53, a versatile pixel detector readout and test system for the ATLAS and CMS HL-LHC upgrades

BDAQ53 is a readout system and verification framework for hybrid pixel detector readout chips of the RD53 family. These chips are designed for the upgrade of the inner tracking detectors of the ATLAS and CMS experiments. BDAQ53 is used in applications where versatility and rapid customization are required, such as in laboratory testing environments, test beam campaigns, and permanent setups for quality control measurements. It consists of custom and commercial hardware, a Python-based software framework, and FPGA firmware. BDAQ53 is developed as open source software with both software and firmware being hosted in a public repository.Comment: 6 pages, 6 figure

Haemoptysis as the first presentation of COVID-19 : a case report

Background Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an ongoing pandemic that profoundly challenges healthcare systems all over the world. Fever, cough and fatigue are the most commonly reported clinical symptoms. Case presentation A 58-year-old man presented at the emergency department with acute onset haemoptysis. On the fifth day after admission, he developed massive haemoptysis. Computed tomography (CT) angiography of the chest revealed alveolar haemorrhage, more prominent in the left lung. Flexible bronchoscopy confirmed bleeding from the left upper lobe, confirmed by a bronchial arteriography, which was successfully embolized. Nasopharyngeal swabs (NPS) tested for SARS-CoV-2 using real-time polymerase chain reaction (RT-PCR) repeatedly returned negative. Surprisingly, SARS-CoV-2 was eventually detected in bronchoalveolar lavage (BAL) fluid. Conclusions Life-threatening haemoptysis is an unusual presentation of COVID-19, reflecting alveolar bleeding as a rare but possible complication. This case emphasises the added value of bronchoscopy with BAL in the diagnostic work-up in case of high clinical suspicion and negative serial NPS in patients presenting with severe symptoms