Hardware Accelerated DNA Sequencing

DNA sequencing technology is quickly evolving. The latest developments ex- ploit nanopore sensing and microelectronics to realize real-time, hand-held devices. A critical limitation in these portable sequencing machines is the requirement of powerful data processing consoles, a need incompatible with portability and wide deployment. This thesis proposes a rst step towards addressing this problem, the construction of specialized computing modules { hardware accelerators { that can execute the required computations in real-time, within a small footprint, and at a fraction of the power needed by conventional computers. Such a hardware accel- erator, in FPGA form, is introduced and optimized specically for the basecalling function of the DNA sequencing pipeline. Key basecalling computations are identi- ed and ported to custom FPGA hardware. Remaining basecalling operations are maintained in a traditional CPU which maintains constant communications with its FPGA accelerator over the PCIe bus. Measured results demonstrated a 137X basecalling speed improvement over CPU-only methods while consuming 17X less power than a CPU-only method

Mitochondrial DNA Analysis by Denaturing High-Performance Liquid Chromatography for the Characterization and Separation of Mixtures in Forensic Samples

A mixture of different mtDNA molecules in a single sample is a significant obstacle to the successful use of standard methods of mtDNA analysis (i.e., dideoxy dye-terminator sequencing). Forensic analysts often encounter either naturally occurring mixtures (e.g., heteroplasmy) or situational mixtures typically arising from a combination of body fluids from separate individuals. The ability to accurately resolve and interpret these types of samples in a timely and cost efficient manner would substantially increase the power of mtDNA analysis and potentially provide valuable investigative information by allowing its use in cases where the current approach is limited or fails. Therefore, this research was aimed at developing a strategy for the use of Denaturing High-Performance Liquid Chromatography (DHPLC) as a developmentally-validated forensic application for resolving mixtures of mtDNA. To facilitate the adoption of this technology by the forensic community, a significant effort has been made to ensure that this technology meets the Scientific Working Group on DNA Analysis Methods (SWGDAM) developmental validation criteria and interfaces smoothly with previously validated methods of forensic mtDNA analysis. To do this, the method developed using DHPLC employs mtDNA amplicons, PCR conditions and DNA sequencing protocols validated for use in forensic laboratories. These factors are essential in implementing DHPLC analysis in a forensic casework environment and for the admissibility of DHPLC and Linkage Phase Analysis in court

Embedded CMOS Basecalling for Nanopore DNA Sequencing

DNA sequencing is undergoing a profound evolution into a mobile technology. Unfortunately the effort needed to process the data emerging from this new sequencing technology requires a compute power only available to traditional desktop or cloud-based machines. To empower the full potential of portable DNA solutions a means of efficiently carrying out their computing needs in an embedded format will certainly be required. This thesis presents the design of a custom fixed-point VLSI hardware implementation of an HMM-based multi-channel DNA sequence processor. A 4096 state (6-mer nanopore sensor) basecalling architecture is designed in a 32-nm CMOS technology with the ability to process 1 million DNA base pairs per second per channel. Over a 100 mm^2 silicon footprint the design could process the equivalent of one human genome every 30 seconds at a power consumption of around 5 W

Advancing the knowledge on the GBA gene and its role on the pathogenesis of Parkinson disease

Since its first description in the XIV century, the understanding of Parkinson disease (PD) has advanced significantly. However, a considerable part of its pathogenesis remains elusive, and no disease modifying therapy has been successfully developed yet and partly because of this lack of knowledge. The genetic background of PD is diverse, accounting for both mendelian, familial and sporadic forms of the disease. One of the main contributors to the genetic risk of PD is the GBA gene, which encodes for a lysosomal enzyme and is also linked to Gaucher disease, an autosomal recessive storage disorder. GBA variants are relatively frequent in sporadic PD, making it a promising target for disease modifying therapies. However, the penetrance of GBA variants is low and mostly unexplained, with only a minority of GBA variants carriers developing PD. In addition, the study of GBA is complicated by difficulties in sequencing the gene, due to the presence of a highly homologous pseudogene (GBAP1) in close proximity to GBA. The aim of this PhD research is to study potential modifiers of risk of PD among GBA variant carriers. First, I collaborated in the development of RAPSODI, an online tool to assess and follow-up a cohort of GBA carriers, both with and without PD, and analysed the data produced, showing interesting differences between GBA carriers and non-carries. The main outcome of this preliminary assessment is that GBA carriers with PD have significantly worse motor and non-motor symptoms compared to GBA-negative PD patients. Further, I refined a method to sequence the GBA gene with Oxford Nanopore’s MinION and developed a novel method for detecting reciprocal recombinants between the gene and pseudogene. By applying these tools, I was able to detect complex structural variants that might modify the risk of PD, as well as explore the role of intronic variants in GBA

On-site identification of animal species in meat products

The control of meat and its products is essential for sales and consumer protection. Due to personal preferences, but also religious and health reasons, the correct labelling is crucial. For meat species inspection, samples have to be sent to a well-equipped laboratory, where a qualified technician extract the DNA from the meat and conduct real-time PCR. The extraction alone takes at least five hours. The real-time polymerase chain reaction (PCR) is used as the gold standard method for the detection of animal species in meat products. The mechanism of real-time PCR is based on three different temperature steps for DNA denaturation (95 °C), primer annealing (60 °C) and elongation (72 °C) of the DNA string. In addition, the use of a fluorescence-labelled probe enables real-time detection of positive signals. In real-time PCR, highly sophisticated, big and expensive devices are required. Furthermore, the run time is around 90 minutes. For the above mentioned reasons, the aim of this doctoral thesis was to determine a rapid detection method for the identification of animal species in meat and meat products in order to simplify on site screening during production or in the sales outlets to enable immediate execution. An isothermal DNA amplification, recombinase polymerase amplification (RPA), was chosen as the detection method. The RPA amplifies its target gene at a constant temperature between 39 and 42 ° C using enzymes and recombinant protein. Similar to the real-time PCR, the successful amplification is visualized by a fluorescence-labelled probe in a maximum of 15 minutes. For the development of the RPA assays for the identification of animal species in meat products, primers and probes were targeting the mitochondrial genes of pork, horse, chicken, turkey, cattle and sheep. The sensitivity of each assay was evaluated by performing eight independent runs using serial concentration of molecular DNA standard of each species (102 to 100 DNA molecules/reaction) and the datasets were subjected to probit-regression analysis. The selected primers were able to amplify their target species with a sensitivity between one and 30 DNA molecules/reaction in a maximum of 11 minutes. No cross-reactions were observed, in other words, each primer combination detects only its target animal species. For field validation of the developed assays, meat and salami mixed samples spiked with various concentration (10, 5, 1, 0.5 and 0.1%) of foreign meat were produced. Each RPA 49 assay was successfully able to detect meat concentration down to of 0.1% in tested samples. The 0.1 % is the lower recommended value by the German Food and Feed Code §64 (LFGB). Moreover, two different fluorescent dyes (FAM and ROX) were applied to detect meat contamination in duplex, whereby one sample can be tested for up to two species in one reaction. No loss of sensitivity in the duplex assays was noticed. This step was important to reduce the assay running costs while maintaining the same productivity. Another important issue in meat industry is the freedom from food borne pathogens. Infectious agents can be ingested through the consumption of contaminated meat and lead to food poisoning in humans. In Africa, however, eating "bush meat" can lead to more contagious and deadly infection like monkey pox (MPXV). In order to diagnose such an infection as early as possible and to start the control measures, the use of a rapid test is beneficial. To make this possible, another RPA assay that detects the monkeypox tumor necrosis factor (TNF) binding protein gene was developed. Both monkey pox clades can be determined with a sensitivity of 16 DNA molecules/μl in 10 minutes. With the selected primer pairs, there is no cross-reaction with the closely related tested viruses or monkey genome. The clinical performance of the MPXV-RPA-assay was tested, revealing a specificity of 100% (50/50), while the sensitivity was 95% (43/45). This assay will pave the way for the identification of food borne infectious agents at low resource settings. Upon presenting my data to the scientific community and end user in international meetings, many individuals have raised the importance of screening more than six animal species. Both RPA and real-time PCR is restricted to the number of the developed assays as well as the fluorescence channels in the detection devices. On other hand, next generation sequencing represents a method with no target limit. For the identification of an unknown adulteration animal source an Oxford nanopore sequencing protocol was tested. The method was combined with offline BLAST search to allow sequencing and data analysis in less than one hour. The developed procedure was successfully detected the contamination of the mock pork sample with 0.1% beef, sheep, goat, horse, donkey, chicken, turkey, duck and rabbit meat. The specificity of the technology was challenged with sequences of exotic animal species as dog, camel, lion, impala, bison and japanese quail. The nanopore sequencing 50 combined with the Offline BLAST search has proven adequate sensitivity and specificity for species identification and represent the future of molecular diagnostics. In summary, in the PhD thesis, not only a rapid on-site detection system for the identification of six animal species in meat products based on the recombinase polymerase amplification were developed, but also a rapid sequencing protocol for the use of the Oxford Nanopore technologies. Both detection methods can be combined with an easy to perform DNA extraction method and all methods can be carried out in a mobile suitcase lab, whereby screening of meat and its products can be performed at point of need. The current work will pave the way for the implementation of such technologies for the benefit of the community and consumers.2021-07-1

Establishing a canine genome sequencing protocol us-ing Oxford Nanopore

The development of sequencing technologies has led to monumental ad-vances in the field of genomics, creating new areas of investigation and profoundly impacting our understanding of life itself. Presently, the “third generation” of these technologies is focused on improving the sequencing of long reads, which allows for studying complex areas in the genome. A promising platform offering long-read sequencing at a comparatively low cost is the Oxford Nanopore Technologies “MinION,” a USB-connected device the size of an ordinary dongle, which can be used in as good as any laboratory setting with a consumer-grade computer. Given that the techno-logy is both recent and still under development, however, there is a need to formulate and verify adequate methodologies for a great variety of target species. In this thesis, a protocol for long-read sequencing of canine DNA using the MinION is presented. Four different HMW-gDNA extraction methods and five library preparation variants were evaluated in order to determine which approach would generate the best sequencing results. Additionally, a method for reusing flow cells in order to maximize data ge-nerated per cell and reducing costs was tested and deemed successful. Major challenges encountered throughout the project include DNA quality, fragment length, as well as high rates of pore loss and low pore occu-pancy. The best-performing DNA extraction protocol was an altered version of Qiagen's Genomic-tip 100/G. For library preparation, a modified version of Nanopore's Sequencing by Ligation kit (SQK-LSK109) had the most favourable results. The best sequencing run generated 14 Gbp of raw data in the span of 48 hours. The results presented herein constitute a first step towards the establishment of a method that leverages the MinION's advan-tages in canine genome sequencing projects.El desarrollo de tecnologías de secuenciación ha conducido a avances monumentales en el campo de la genómica, creando nuevas áreas de in-vestigación e impactando profundamente nuestro entendimiento de la vida misma. Actualmente, la "tercera generación" de estas tecnologías se con-centra en mejorar la secuenciación de lecturas largas, lo que permite estu-diar áreas complejas del genoma. Una nueva y prometedora plataforma que ofrece secuenciación de lecturas largas a un costo comparativamente bajo es el “MinION”, de la compañía Oxford Nanopore Technologies, cuyo tamaño, similar al de un adaptador USB, permite que pueda utilizarse en cualquier tipo de laboratorio. Sin embargo, dado que esta tecnología es relativamente reciente y aún se encuentra en desarrollo, es necesario formu-lar nuevas metodologías que sean adecuadas para diferentes tipos de especies. Esta tesis presenta un protocolo para la secuenciación de lectu-ras largas de ADN canino utilizando el dispositivo MinION. Se evaluaron cuatro métodos de extracción de ADN de alto peso molecular y cinco mé-todos de preparación de bibliotecas con el fin de determinar qué protocolo produce los mejores resultados. Asimismo, con el fin de maximizar los datos generados por celda de flujo y reducir costos, se analizó un método para reutilizar celdas de flujo, el cual fue considerado exitoso. Los principa-les desafíos que se encontraron a lo largo de este proyecto incluyen la obtención de ADN de calidad y de alto peso molecular, así como la alta tasa de pérdida de nanoporos. El protocolo de extracción de ADN que produjo los mejores resultados fue una versión alterada del kit de Qiagen Genomic-tip 100/G. Para la preparación de la biblioteca, una versión modi-ficada del kit de Secuenciación por Ligadura de Nanopore (SQK-LSK109) tuvo los resultados más favorables. El mejor experimento de secuencia-ción generó 14 Gbp en el lapso de 48 horas. Los resultados aquí presenta-dos constituyen un primer paso para el establecimiento de un método que aprovecha las ventajas del MinION para proyectos de secuenciación del genoma canino

Practical Tools to Implement Massive Parallel Pyrosequencing of PCR Products in Next Generation Molecular Diagnostics

Despite improvements in terms of sequence quality and price per basepair, Sanger sequencing remains restricted to screening of individual disease genes. The development of massively parallel sequencing (MPS) technologies heralded an era in which molecular diagnostics for multigenic disorders becomes reality. Here, we outline different PCR amplification based strategies for the screening of a multitude of genes in a patient cohort. We performed a thorough evaluation in terms of set-up, coverage and sequencing variants on the data of 10 GS-FLX experiments (over 200 patients). Crucially, we determined the actual coverage that is required for reliable diagnostic results using MPS, and provide a tool to calculate the number of patients that can be screened in a single run. Finally, we provide an overview of factors contributing to false negative or false positive mutation calls and suggest ways to maximize sensitivity and specificity, both important in a routine setting. By describing practical strategies for screening of multigenic disorders in a multitude of samples and providing answers to questions about minimum required coverage, the number of patients that can be screened in a single run and the factors that may affect sensitivity and specificity we hope to facilitate the implementation of MPS technology in molecular diagnostics