Biological variations are being explored at ever-increasing rates through the rapid advancement of analytical techniques. Techniques like massively parallel sequencing empower scientists to accurately differentiate individuals’ genetic compositions, cellular functionalities, and healthy tissue from diseased. The knowledge gained from these techniques brings us ever closer to grasping the complexities of life, contributing to human development. Still, to fully elucidate biological variations in different samples requires novel sensitive and high- throughput techniques, capable of placing everything in its correct context. One such technique gaining promise is droplet barcoding. 

Droplet barcoding leverages emulsion droplets to segregate samples into their functional components, coupled with barcodes that can group tagged molecules following sequencing. This technique constitutes a versatile tool for studying biological variations in both the phenotype and genotype. This thesis leverages droplet barcoding to explore variations relating to human biology. 

Droplet barcoding was used to study phenotype variations, looking at protein compositions in single extracellular vesicles (Paper I) and single cells (Paper II). Paper I studies extracellular vesicles which are naturally released from cells. They carry heterogeneous protein signatures that can inform about their cellular origin. Tens of thousands of extracellular vesicles were profiled, including approximately 25,000 from lung cancer patients. From these protein profiles, extracellular vesicles could be grouped into putative subtypes. Paper II presents a novel method for studying single cells which was used to characterize blood-derived immune cells. The method enabled the identification of most major immune cell lineages. 

Haplotype-resolved genetic variations were analyzed using a linked read sequencing method based on droplet barcoding. Linked-read sequencing conserves long-range information from short-read sequencing by co- barcoding subsections of long DNA fragments. Paper III presents an open-source pipeline (BLR) for whole genome haplotyping using linked reads. BLR generates accurate and continuous haplotypes, outperforming PacBio HiFi-based diploid assembly. We further show that integration with low-coverage long-read data can improve phasing accuracy in tandem repeats. With 10X Genomics linked reads, BLR generated more continuous haplotypes compared to other workflows. Paper IV applies linked read sequencing to reveal the haplotype complexities of cancer genomes. In two patients with colorectal cancer, we identified several large-scale aberrations impacting cancer-related genes. Additionally, several short somatic variants were found to impact nearly all oncogenic networks identified by TCGA. Demonstrating the importance of haplotype-resolved analysis for cancer genomics, one patient exhibited two nonsense mutations on separate haplotypes in the well-known colorectal cancer gene APC. 

Biologiska variationer utforskas i allt snabbare grad, pådrivet av den snabba utvecklingen av analytiska tekniker. Tekniker som massiv parallellsekvensering möjliggör för forskare att noggrant särskilja individers genetiska sammansättningar, cellernas olika funktioner och frisk vävnad från sjuk. Vetskapen dessa tekniker medför ger oss allt djupare insikter om livsformers komplexitet som främjar mänsklig utveckling. Torts dessa framsteg kräver klarläggandet av biologiska variationer i olika prover nya känsliga tekniker med hög kapacitet, kapabla att placera information i dess rätta sammanhang. En särskilt lovande teknik är droppkodning. 

Droppkodning utnyttjar emulsionsdropparnas förmåga att separera prover i dess funktionella komponenter kombinerat med DNA-koder för att gruppera märkta molekyler efter sekvensering. Denna teknik utgör ett mångsidigt verktyg för att studera biologiska variationer i både fenotyp och genotyp. Den här avhandlingen utforskar tekniker baserat på droppkodning för att analysera dessa variationer relaterat till människlig biologi. 

Droppkodning användes i analys av fenotypvariationer genom att studera proteinsignaturer hos enskilda extracellulära vesiklar (Artikel I) samt enskilda celler (Artikel II). Artikel I studerar extracellulära vesiklar, vilka är partiklar som naturligt släpps ut från celler. Dessa vesiklar bär på heterogena protein-signaturer som kan informera om dess cellulära härkomst. I studien undersöks proteinsignaturer från tiotusentals extracellulära vesiklar, inklusive cirka 25 000 från lungcancerpatienter. Utifrån dessa signaturer kunde extracellulära vesiklar sedan grupperas i potentiella subtyper. Artikel II presenterar en ny metod för att studera enskilda celler, som användes för att karakterisera immunceller från blod. Metoden möjliggjorde identifiering av de flesta stora immuncellspopulationerna. 

Haplotyp-upplösta genotypvariationer analyserades med en metod för länkad sekvensering baserad på droppkodning. Länkad sekvensering möjliggör att vid sekvensering med kort läslängd bevara information över långa genomiska distanser genom DNA-kodning av små delar av långa DNA-fragment. Artikel III presenterar en pipeline (BLR) med öppen källkod för helgenoms haplotypning som använder data från länkad sekvensering. BLR genererar haplotyper med stor exakthet och kontinuitet som överträffar diploid genom-sammansättning (“assembly”) med PacBio HiFi data. Vi visar även att integrering med långa sekvenser med begränsad genomtäckning förbättra haplotypning i tandem-repetitiva genomregioner. Med 10X Genomics länkade sekvenser genererade BLR mer kontinuerlig haplotypning jämfört med andra analysflöden. Artikel IV tillämpar länkad sekvensering för att avslöja haplotypkomplexiteten hos cancergenom. Hos två patienter med tjocktarmscancer identifierades flera storskaliga variationer som överlappar cancerrelaterade gener. Dessutom hittades flera korta somatiska varianter som påverkade gener i nästan all onkogena nätverk identifierade av TCGA. En patient uppvisade två nonsensmutationer på separata haplotyper i den välkända tjocktarmscancergenen APC, vilket påvisar vikten av haplotyp-upplöst analys för cancergenomik. 

Linked-read sequencing promises a one-method approach for genome-wide insights including single nucleotide variants (SNVs), structural variants, and haplotyping. We introduce Barcode Linked Reads (BLR), an open-source haplotyping pipeline capable of handling millions of barcodes and data from multiple linked-read technologies including DBS, 10× Genomics, TELL-seq and stLFR. Running BLR on DBS linked-reads yielded megabase-scale phasing with low (<0.2%) switch error rates. Of 13616 protein-coding genes phased in the GIAB benchmark set (v4.2.1), 98.6% matched the BLR phasing. In addition, large structural variants showed concordance with HPRC-HG002 reference assembly calls. Compared to diploid assembly with PacBio HiFi reads, BLR phasing was more continuous when considering switch errors. We further show that integrating long reads at low coverage (∼10×) can improve phasing contiguity and reduce switch errors in tandem repeats. When compared to Long Ranger on 10× Genomics data, BLR showed an increase in phase block N50 with low switch-error rates. For TELL-Seq and stLFR linked reads, BLR generated longer or similar phase block lengths and low switch error rates compared to results presented in the original publications. In conclusion, BLR provides a flexible workflow for comprehensive haplotype analysis of linked reads from multiple platforms.

Small extracellular vesicles (sEVs) have in recent years evolved as a source of biomarkers for disease diagnosis and therapeutic follow up. sEV samples derived from multicellular organisms exhibit a high heterogeneous repertoire of vesicles which current methods based on ensemble measurements cannot capture. In this work we present droplet barcode sequencing for protein analysis (DBS-Pro) to profile surface proteins on individual sEVs, facilitating identification of sEV-subtypes within and between samples. The method allows for analysis of multiple proteins through use of DNA barcoded affinity reagents and sequencing as readout. High throughput single vesicle profiling is enabled through compartmentalization of individual sEVs in emulsion droplets followed by droplet barcoding through PCR. In this proof-of-concept study we demonstrate that DBS-Pro allows for analysis of single sEVs, with a mixing rate below 2%. A total of over 120,000 individual sEVs obtained from a NSCLC cell line and from malignant pleural effusion (MPE) fluid of NSCLC patients have been analyzed based on their surface proteins. We also show that the method enables single vesicle surface protein profiling and by extension characterization of sEV-subtypes, which is essential to identify the cellular origin of vesicles in heterogenous samples.

Background: Hundreds of variants associated with atopic dermatitis (AD) and psoriasis, 2 common inflammatory skin disorders, have previously been discovered through genome-wide association studies (GWASs). The majority of these variants are in noncoding regions, and their target genes remain largely unclear. Objective: We sought to understand the effects of these noncoding variants on the development of AD and psoriasis by linking them to the genes that they regulate. Methods: We constructed genomic 3-dimensional maps of human keratinocytes during differentiation by using targeted chromosome conformation capture (Capture Hi-C) targeting more than 20,000 promoters and 214 GWAS variants and combined these data with transcriptome and epigenomic data sets. We validated our results with reporter assays, clustered regularly interspaced short palindromic repeats activation, and examination of patient gene expression from previous studies. Results: We identified 118 target genes of 82 AD and psoriasis GWAS variants. Differential expression of 58 of the 118 target genes (49%) occurred in either AD or psoriatic lesions, many of which were not previously linked to any skin disease. We highlighted the genes AFG1L, CLINT1, ADO, LINC00302, and RP1-140J1.1 and provided further evidence for their potential roles in AD and psoriasis. Conclusions: Our work focused on skin barrier pathology through investigation of the interaction profile of GWAS variants during keratinocyte differentiation. We have provided a catalogue of candidate genes that could modulate the risk of AD and psoriasis. Given that only 35% of the target genes are the gene nearest to the known GWAS variants, we expect that our work will contribute to the discovery of novel pathways involved in AD and psoriasis.

Profiling the genome-wide chromatin accessibility via Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) provides important insights into genome regulation that help understanding different biological processes. However, the heterogeneity of cell populations must be considered to gain a full understanding which is possible through droplet microfluidics enabling single-nuclei ATAC-seq (snATAC-seq). Droplet microfluidic platforms commonly used to perform snATAC-seq lack versatility and cannot be easily adapted to custom assay requirements. Here, we present a fully versatile, low-cost droplet microfluidic platform that can be readily adapted to run various droplet-based library preparation protocols for analyses at single-cell resolution such as snATAC-seq. 

BACKGROUND: Genetic variant landscape of coronary artery disease is dominated by noncoding variants among which many occur within putative enhancers regulating the expression levels of relevant genes. It is crucial to assign the genetic variants to their correct genes both to gain insights into perturbed functions and better assess the risk of disease. METHODS: In this study, we generated high-resolution genomic interaction maps (similar to 750 bases) in aortic endothelial, smooth muscle cells and THP-1 (human leukemia monocytic cell line) macrophages stimulated with lipopolysaccharide using Hi-C coupled with sequence capture targeting 25 429 features, including variants associated with coronary artery disease. We also sequenced their transcriptomes and mapped putative enhancers using chromatin immunoprecipitation with an antibody against H3K27Ac. RESULTS: The regions interacting with promoters showed strong enrichment for enhancer elements and validated several previously known interactions and enhancers. We detected interactions for 727 risk variants obtained by genome-wide association studies and identified novel, as well as established genes and functions associated with cardiovascular diseases. We were able to assign potential target genes for additional 398 genome-wide association studies variants using haplotype information, thereby identifying additional relevant genes and functions. Importantly, we discovered that a subset of risk variants interact with multiple promoters and their expression levels were strongly correlated. CONCLUSIONS: In summary, we present a catalog of candidate genes regulated by coronary artery disease-related variants and think that it will be an invaluable resource to further the investigation of cardiovascular pathologies and disease.

Proteins play a pivotal role in cellular function and heterogeneity. Understanding cellular diversity at the proteome level necessitates sensitive single-cell assays with high throughput. While current sequencing-based methods offer promise, they often face limitations, including reliance on expensive and inaccessible commercial platforms. Here, we have adopted the DBS-Pro method, utilizing site-specific oligonucleotide-conjugated antibodies, to analyze surface proteins in single cells. The method uses cheap degenerated barcode oligonucleotides and a simple microfluidics setup for cell encapsulation. A sample of PBMCs was examined using a panel targeting six separate immune cell markers. Using this panel we could quantify marker expression on 1,307 cells, identifying major immune cell lineages including CD4+ T-cells, CD8+ T-cells, monocytes, and B-cells. While recognizing the need for protocol improvements, our results present a promising approach for single-cell proteomics.

Cancer genomes are prone to elevated rates of genomic alterations. Massive parallel sequencing technologies can answer some questions related to these aberrations; however, they remain limited when it comes to resolving the haplotype information. In this study, we applied the linked-read droplet barcode sequencing (DBS) technology to resolve the haplotype complexity of colorectal cancer genomes, using paired tumor/normal samples. The results show short somatic variants associated with almost all TCGA-identified oncogenic pathways. Several cancer-related genes had multiple variants in either one or both haplotypes. In the tumor suppressor gene APC, two nonsense variants ~2kb apart on separate haplotypes were identified in one patient. Additionally, a number haplotype-resolved somatic structural variants (SV) and copy number alterations (CNA) were detected and correlated with the small variants. The study demonstrates that DBS technology can characterize complex genetic variations in a haplotype context, revealing an extra layer of cancer genome complexity.