High-throughput chromatin accessibility profiling at single-cell resolution.

Here we develop a high-throughput single-cell ATAC-seq (assay for transposition of accessible chromatin) method to measure physical access to DNA in whole cells. Our approach integrates fluorescence imaging and addressable reagent deposition across a massively parallel (5184) nano-well array, yielding a nearly 20-fold improvement in throughput (up to ~1800 cells/chip, 4-5 h on-chip processing time) and library preparation cost (~81¢ per cell) compared to prior microfluidic implementations. We apply this method to measure regulatory variation in peripheral blood mononuclear cells (PBMCs) and show robust, de novo clustering of single cells by hematopoietic cell type

Landscape of stimulation-responsive chromatin across diverse human immune cells.

A hallmark of the immune system is the interplay among specialized cell types transitioning between resting and stimulated states. The gene regulatory landscape of this dynamic system has not been fully characterized in human cells. Here we collected assay for transposase-accessible chromatin using sequencing (ATAC-seq) and RNA sequencing data under resting and stimulated conditions for up to 32 immune cell populations. Stimulation caused widespread chromatin remodeling, including response elements shared between stimulated B and T cells. Furthermore, several autoimmune traits showed significant heritability in stimulation-responsive elements from distinct cell types, highlighting the importance of these cell states in autoimmunity. Allele-specific read mapping identified variants that alter chromatin accessibility in particular conditions, allowing us to observe evidence of function for a candidate causal variant that is undetected by existing large-scale studies in resting cells. Our results provide a resource of chromatin dynamics and highlight the need to characterize the effects of genetic variation in stimulated cells

Padlock Probe-Based Assays for Molecular Diagnostics

Treatment success often depends on the availability of accurate and reliable diagnostic assays to guide clinical practitioners in their treatment choices. An optimal test must excel in specificity and sensitivity, and depending on the application area time, low-cost and simplicity are equally important. For instance, time is essential in infectious diagnostics but this is less important in non-invasive prenatal testing (NIPT). In NIPT, specificity and sensitivity are the most important parameters. In this thesis I describe the development of four different methods, all based on padlock probes and rolling circle amplification, intended for molecular diagnostics. Application areas range from infectious disease diagnostics to NIPT and oncology. The methods described have in common that they overcome certain limitations of currently available assays. This thesis includes two new assays targeting infectious agents: one assay specifically detecting a highly variable double stranded RNA virus and the second assay demonstrating a new format of antibiotic susceptibility testing, which is rapid and generally applicable to different pathogens. Furthermore, I describe the development of a method that uses methylation markers to enrich fetal DNA, accurately quantify chromosome ratios and thus, detecting trisomy 21 and 18. The fourth method described in this thesis uses gap-fill ligation of padlock probes to detect diagnostic relevant point mutations with high specificity in situ. The assays presented have the potential, after automation and successful validation and verification studies, to be implemented into clinical practice. Furthermore, these assays demonstrate the wide applicability of padlock probes which, due to their properties in regard to specificity and multiplexity, are useful tools for nucleic acid detection in vitro as well as in situ.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p

Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification

Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ.

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ

Analysis of patient samples.

<p>cDNA was prepared from 22 patient samples collected at Uppsala University hospital. (A) PCR positive samples. (B) PCR negative samples. Plotted is the number of rolling circle product - (negative+3 SD) on the y-axis and patient samples on the x-axis. Error bars ±1 s.d.; n = 2.</p