Tissue structure and molecular circuitry in the colon can be profoundly impacted by systemic age-related effects but many of the underlying molecular cues remain unclear. Here, we build a cellular and spatial atlas of the colon across three anatomical regions and 11 age groups, encompassing similar to 1,500 mouse gut tissues profiled by spatial transcriptomics and similar to 400,000 single nucleus RNA-sequencing profiles. We develop a computational framework, cSplotch, which learns a hierarchical Bayesian model of spatially resolved cellular expression associated with age, tissue region and sex by leveraging histological features to share information across tissue samples and data modalities. Using this model, we identify cellular and molecular gradients along the adult colonic tract and across the main crypt axis and multicellular programs associated with aging in the large intestine. Our multimodal framework for the investigation of cell and tissue organization can aid in the understanding of cellular roles in tissue-level pathology.

Mucosal and barrier tissues, such as the gut, lung or skin, are composed of a complex network of cells and microbes forming a tight niche that prevents pathogen colonization and supports host–microbiome symbiosis. Characterizing these networks at high molecular and cellular resolution is crucial for understanding homeostasis and disease. Here we present spatial host–microbiome sequencing (SHM-seq), an all-sequencing-based approach that captures tissue histology, polyadenylated RNAs and bacterial 16S sequences directly from a tissue by modifying spatially barcoded glass surfaces to enable simultaneous capture of host transcripts and hypervariable regions of the 16S bacterial ribosomal RNA. We applied our approach to the mouse gut as a model system, used a deep learning approach for data mapping and detected spatial niches defined by cellular composition and microbial geography. We show that subpopulations of gut cells express specific gene programs in different microenvironments characteristic of regional commensal bacteria and impact host–bacteria interactions. SHM-seq should enhance the study of native host–microbe interactions in health and disease.

The spatial organization of cells and molecules plays a key role in tissue function in homeostasis and disease. Spatial transcriptomics has recently emerged as a key technique to capture and positionally barcode RNAs directly in tissues. Here, we advance the application of spatial transcriptomics at scale, by presenting Spatial Multi-Omics (SM-Omics) as a fully automated, high-throughput all-sequencing based platform for combined and spatially resolved transcriptomics and antibody-based protein measurements. SM-Omics uses DNA-barcoded antibodies, immunofluorescence or a combination thereof, to scale and combine spatial transcriptomics and spatial antibody-based multiplex protein detection. SM-Omics allows processing of up to 64 in situ spatial reactions or up to 96 sequencing-ready libraries, of high complexity, in a similar to 2 days process. We demonstrate SM-Omics in the mouse brain, spleen and colorectal cancer model, showing its broad utility as a high-throughput platform for spatial multi-omics.

Novel insights into biological functions and mechanisms, cell networks and evolutionary relationships are gained through development of sequencing technologies and sequencing based applications. Massively parallel sequencing has enabled analysis of big data at gene and protein expression levels, but has also characterized bacterial communities. Additionally, different technological advancements enabled us to track those expression changes in single cells, to reveal insights into rare cell populations, or with added spatial resolution, to explore highly complex environments such as tissues. This thesis gives an overview of different technical, biological and computational methods used in genomics today with a specific focus on spatial techniques for detailed tissue characterization. This is followed by a chapter summarizing recent scientific contributions made by the author that have been included as part of this thesis. In Paper I, 16S sequencing was used to study the diversity and composition of bacterial communities with specific focus on the aerodigestive microbiome in children who had undergone a lung transplant. Potential connections between the microbiome and irregular gastric muscle movements were also examined. Patients with a lung transplant had significantly lower microbial diversity in the gastric and oropharyngeal sites as compared to controls, however, lung transplant recipients showed similar bacterial compositions, independent of motility status. Samples in the lung transplant patient group were in general dominated by Staphylococcaceae but Streptococcus, Prevotella and Veillonella were common in the gastric and oropharyngeal samples. Next, an automated method for simultaneous spatial analysis of both gene and antibodybased protein expression in tissue sections, named SM-Omics, was developed in Paper II. SM-Omics enabled simultaneous detection of proteins, by using either immunofluorescence or DNAbarcoded antibodies, and analysis of the spatial transcriptome in the same tissue section. SM-Omics was applied to the mouse brain and spleen and obtained correlated spatial patterns between respective gene and antibody measurements. The method allowed processing of up to 64 in situ spatial reactions or up to 96 sequencing-ready libraries, of high complexity, in a ~2 days process. The spatial host-microbiome sequencing method, presented in Paper III, was used to concurrently study the spatial environment created between bacteria and host cells within a tissue section. Using spatial host-microbiome sequencing, colonic sections from three different mouse models were examined by simultaneous in situ capture of both mRNA and 16S sequences, followed by sequencing and taxonomic assignment of bacterial 16S sequences using a deep learning model. ~17,000 genes and 39 bacteria genera across 16 different morphological regions were quantitatively assessed in the mouse colon. We reported specific genera in the interfold and lumen regions of the colon, as well as spatially variable genes across 100 tissue sections. To better understand genotype-relevant changes impacted by bacterial presence, we defined cell-type specific interactions described with sets of activated pathways. Finally, consecutive tissue sections of multiple synovial biopsies from patients suffering from rheumatoid arthritis were processed using the Spatial Transcriptomics method and sequenced in Paper IV. The alignment and transformation of the consecutive tissue sections enabled spatial profiling in 3D of genes and cell types within the biopsies. Spatially variable gene expression patterns revealed clusters radially distributed around organized structures of infiltrating leukocytes (TLOs). In patients with developed TLOs, these structures contained proinflammatory B cells, while the surrounding areas were high in fibroblasts.

Utvecklingen av sekvensering-teknologier och applikationer som baseras på sekvensering, har lett till ny förståelse av biologiska funktioner och mekanismer, cell-nätverk och evolutionära samband. Massively parallel sequencing har möjliggjort analys av big data som behandlar gen och proteinuttryck, men också för karakterisering av bakteriella samhällen. Olika tekniska framsteg har dessutom tillåtit oss att studera dessa uttryck i individuella celler, få en ökad förståelse av sällsynta cellpopulationer och adderat den rumslig upplösningen, för att undersöka mycket komplexa miljöer såsom vävnader. Den här avhandlingen börjar med en överblick av olika tekniska, biologiska och datorbaserade metoder som används inom genomik idag, med ett speciellt fokus på tekniker som genererar en rumslig upplösning för detaljerad beskrivning av vävnader. Efter detta följer ett kapitel som summerar författarens senaste vetenskapliga bidrag som är en del av denna avhandling. I Artikel I användes 16S-sekvensering för att studera diversitet och sammansättning av bakteriella samhällen. Fokus var på mikrobiomet i luft- och matspjälkningskanalen hos barn med lungtransplantation. Potentiella samband mellan mikrobiomet och störningar i mag-tarmkanalens rörelser studerades också. Patienter med lungtransplantation hade signifikant lägre mikrobiell diversitet i magen och halsen. Dock visade sig denna grupp även ha liknande bakteriell sammansättning, oberoende av mag-tarmkanalens rörelser. Patienterna dominerades av Staphylococcaceae men Streptococcus, Prevotella och Veillonella var vanligt förekommande i magen och halsen. Vidare presenteras utvecklingen av SM-Omics i Artikel II, en automatiserad metod för simultan rumslig analys av både gener och antikroppsbaserat proteinuttryck i vävnadssnitt. SM-Omics möjliggjorde simultan detektion av proteiner, genom att använda antingen immunofluorescens eller DNA-märkta antikroppar, och analys av den rumsliga upplösningen av transkriptomet i samma vävnadssnitt. SM-Omics applicerades på mushjärna och musmjälte och uppnådde korrelerade rumsliga mönster mellan respektive gen- och protein-uttryck och DNAmärkta antikroppsmätningar. Metoden möjliggör framställning av upp till 64 in situ-reaktioner med rumslig upplösning eller upp till 96 sekvenseringsbara bibliotek, av hög komplexitet, på ~2 dagar. Spatial host-microbiome sequencing, som presenteras i Artikel III, är en metod som används för att studera den rumsliga miljön som skapats mellan bakterier och värdceller inuti ett vävnadssnitt. Genom användning av Spatial host-microbiome sequencing kunde simultan in situinfångning av både mRNA och 16S-sekvenser från vävnadssnitt av tjocktarmen från tre olika musmodeller erhållas och sekvenseras. En deep learning-modell användes för att taxonomiskt identifiera 16S-sekvenserna. ~17000 gener och 39 bakteriella släkten var kvantitativt analyserade i 16 olika morfologiska regioner i mustarmen. Vi identifierade specifika släkten i tarmens hålrum och rumsliga variationer i genuttrycket i 100 vävnadssnitt. För att bättre förstå eventuell bakteriell påverkan på genotypiska skillnader, definierade vi celltyps-specifika interaktioner genom att beskriva aktiverade nätverk. Slutligen, konsekutiva vävnadssnitt från multipla biopsier av synovialled från patienter med ledgångsreumatism var preparerade med Spatial Transcriptomics-metoden och sekvenserade i Artikel IV. Justering och transformation av de konsekutiva vävnadssnitten möjliggjorde rumslig profilering i 3D av gener och celltyper inom biopsierna. Rumsliga variationer i genuttrycket påvisade radiellt distribuerade kluster runt organiserade strukturer med infiltrerande vita blodkroppar (TLOs). Hos patienter med utvecklade TLOs, innehöll dessa strukturer proinflammatoriska B-celler, medan omgivande områden innehöll många fibroblaster.

The inflamed rheumatic joint is a highly heterogeneous and complex tissue with dynamic recruitment and expansion of multiple cell types that interact in multifaceted ways within a localized area. Rheumatoid arthritis synovium has primarily been studied either by immunostaining or by molecular profiling after tissue homogenization. Here, we use Spatial Transcriptomics, where tissue-resident RNA is spatially labeled in situ with barcodes in a transcriptome-wide fashion, to study local tissue interactions at the site of chronic synovial inflammation. We report comprehensive spatial RNA-Seq data coupled to cell type-specific localization patterns at and around organized structures of infiltrating leukocyte cells in the synovium. Combining morphological features and high-throughput spatially resolved transcriptomics may be able to provide higher statistical power and more insights into monitoring disease severity and treatment-specific responses in seropositive and seronegative rheumatoid arthritis. Sanja Vickovic et al. use spatial transcriptomics to probe the local synovial tissue interactions in rheumatoid arthritis (RA) patients. Their results provide a valuable resource to understand the spatial organisation of cell populations in the synovium in the context of RA-associated inflammation.

BACKGROUND: Delayed gastric emptying has been associated with increased graft rejection, although the mechanism of this association is not known. This study aims to investigate the interrelationship between delays in gastrointestinal motility and the diversity and composition of gastric, oropharyngeal, and lung microbiomes in pediatric lung transplant recipients. METHODS: We prospectively recruited 23 pediatric lung transplant recipients and 98 pediatric patients with respiratory symptoms undergoing combined endoscopy and bronchoscopy. Gastric, oropharyngeal, and bronchoalveolar lavage samples were collected for 16S sequencing. Gastric samples were also analyzed for bile composition using liquid chromatography. RESULTS: Patients who underwent lung transplantation had significantly reduced alpha diversity in gastric and oropharyngeal sites compared with patients with respiratory symptoms. This reduction in alpha diversity was especially evident in gastric samples in patients with delayed gastric emptying defined as abnormal gastric emptying on nuclear scintigraphy or as an elevation in gastric bile concentration (p ≤ 0.05). Whereas monocolonies were seen in the lungs of patients who underwent transplantation, these were not the same microbes seen in the stomach; the microbial overlap between lung and gastric samples within patients was low, and data indicated high individual variation between lung transplant recipients. Other contributors to reduced alpha diversity included antibiotics in combination with proton pump inhibitors, especially in gastric and oropharyngeal samples. CONCLUSIONS: Lung transplant recipients have reduced microbial diversity in gastric fluid (GF) and oropharynx compared with patients who did not undergo lung transplantation. The decreased alpha diversity in GF may be associated with dysmotility.

Sequencing technologies and applications have pushed the limits and enabled novel studies of biological mechanisms, evolutionary relationships and communication networks between cells. The technical developments leading to single cell RNA-sequencing have enabled detection of rare cell populations while spatial resolution added insights into larger biological environments, like tissues and organs. Massively parallel sequencing has paved the way for integrated high-throughput analyses including that of studying gene expression, protein expression and mapping of microbial communities. This thesis starts with an introduction describing the technical and biological advancements made in recent years with focus on spatially resolved approaches. Then, a summary of recent accomplishments is presented, which enabled ongoing work in a novel field of spatial hostmicrobiome profiling. Lastly, the concluding remarks include both a future perspective and a short reflection on the current developments in the spatial multi-omics field. 16S sequencing is often used for taxonomic classification of bacteria. In Paper I, this sequencing technique was used to study the aerodigestive microbiome in pediatric lung transplant recipients. Many of these patients regretfully reject the organ after transplant, but the underlying cause is, in many cases, unknown. In this paper, multiple factors influencing rejection were examined including that of the aerodigestive microbiome. Pediatric lung transplant recipients often suffer from gastrointestinal dysmotility and the focus of this study was also to analyze changes in the microbiome in relation to irregular gastric muscle movements. The results showed that lung transplant recipients had, in general, lower microbial diversity in the gastric fluid and throat and also that the microbial overlap between lung and gastric sampling sites was significantly less in transplant recipients compared to controls. In addition, gastrointestinal dysmotility was shown to influence the gastric microbiome in lung transplant recipients, but, given the small sample size available in this study, the correlation to patient outcome could not be examined. Integrated analysis of the transcriptome and the antibody-based proteome in the same tissue section was enabled using the method developed in Paper II. Spatial Multi- Omics (SM-Omics) uses a barcoded glass array to capture mRNA and antibody-based expression of selected proteins in the same section. The antibody-based profiling of the tissue section was enabled by either immunofluorescence or DNA-barcoded antibodies that were then decoded by sequencing. The protocol was scaled-up using an automated liquidhandling system. Using this method, simultaneous profiling of the transcriptome and multiplexed protein values was determined in both the mouse brain cortex and mouse spleen. Results showed a high correlation in spatial pattern between gene expression and antibody measurements, independently of the antibody labelling technique. SM-Omics generates a high-plex multi-omics characterization of the tissue in a high throughput manner while exhibiting low technical variation.

Tekniker och applikationer som använder sekvensering har flyttat fram gränsernaoch tillåtit nya undersökningar av biologiska mekanismer, evolutionära släktskap ochkommunikationsnätverk mellan celler. De tekniska utvecklingarna som har lett fram tillRNA-sekvensering av enskilda celler har möjliggjort upptäckten av sällsynta cellpopulationer medan den rumsliga upplösningen har inneburit en ökad förståelse av störrebiologiska miljöer, såsom vävnader och organ. Massively parallel sequencing har banat vägför integrerade analyser med hög kapacitet, vilket inkluderar analys av genuttryck,proteinuttryck och kartläggning av bakteriella samhällen. Den här avhandlingen börjar meden introduktion som beskriver tekniska och biologiska framsteg som gjorts de senaste åren,med fokus på den rumsliga upplösningen. Sedan följer en summering av de senasteprestationerna som har möjliggjort det pågående arbetet i ett nytt fält som avhandlarrumslig profilering av bakterien och dess värd. Slutligen innehåller slutordet både ettframtida perspektiv samt en kort reflektion av den nuvarande utvecklingen inom fälten förrumslig mång-omik.

16S-sekvensering används ofta för att taxonomiskt klassificera bakterier. Dennasekvenseringsteknik användes i artikel I för att studera mikrobiomet i luft- ochmatspjälkningskanalen hos barn med transplanterad lunga. Dessvärre är det vanligt medavstötning av lungan efter transplantationen hos många av dessa patienter, men denunderliggande orsaken till avstötningen är, i många fall, okänd. I denna studie undersöktesflertalet faktorer, inklusive mikrobiomet i luft- och matspjälkningskanalen, som kan tänkaspåverka bortstötningen. Barn med transplanterad lunga lider ofta av störningar i magtarmkanalens rörelser och artikelns fokus var därmed även att analysera förändringar imikrobiomet i relation till dessa avvikande rörelser i mag-tarmkanalen. Resultatet visade attpatienter med transplanterad lunga generellt hade lägre bakteriell mångfald i magsaft ochhals, samt att det bakteriella överlappet mellan lunga och magsaft var signifikant mindre ipatienter med transplanterad lunga jämfört med kontrollerna. För övrigt visade det sig attstörningar i mag-tarmkanalens rörelser påverkade magsaftens mikrobiom hos patientermed transplanterad lunga, men på grund av studiens storlek på urvalet, kunde det inteundersökas hur detta korrelerade till utfallet hos patienterna.

Integrerad analys av transkriptomet och antikroppsbaserad analys av proteomet isamma vävnadssnitt har möjliggjorts genom metoden som utvecklats i artikel II. SpatialMulti-Omics (SM-Omics) använder ett avkodningsbart mönster av korta DNA-segment påen glasyta för att fånga mRNA och antikroppsbaserat uttryck av utvalda proteiner frånsamma vävnadssnitt. Den antikroppsbaserade profileringen av vävnadssnittet uppnåddesgenom antingen immunofluorescens eller antikroppar märkta med DNA-segment somkunde avkodas genom sekvensering. Protokollet skalades upp genom ett automatiseratsystem för att behandla vätskor. Genom användning av denna metod kunde simultanprofilering av transkriptomet och flertalet proteiner uppnås i både hjärnbarken och mjältenhos en mus. Resultaten visade en hög korrelation i det rumsliga mönstret mellangenuttrycket och de antikroppsbaserade mätningarna, oberoende av hur antikropparnahade märkts. SM-Omics genererar en storskalig karaktärisering av vävnaden av flera omikermed hög kapacitet samtidigt som den har låg teknisk variation.

BACKGROUND:Delayed gastric emptying has been associated with increased graft rejection, althoughthe mechanism of this association is not known. This study aims to investigate the interrelationshipbetween delays in gastrointestinal motility and the diversity and composition of gastric, oropharyngeal,and lung microbiomes in pediatric lung transplant recipients.METHODS:We prospectively recruited 23 pediatric lung transplant recipients and 98 pediatric patientswith respiratory symptoms undergoing combined endoscopy and bronchoscopy. Gastric, oropharyn-geal, and bronchoalveolar lavage samples were collected for 16S sequencing. Gastric samples werealso analyzed for bile composition using liquid chromatography.RESULTS:Patients who underwent lung transplantation had significantly reduced alpha diversity in gas-tric and oropharyngeal sites compared with patients with respiratory symptoms. This reduction in alphadiversity was especially evident in gastric samples in patients with delayed gastric emptying defined asabnormal gastric emptying on nuclear scintigraphy or as an elevation in gastric bile concentration (p≤0.05). Whereas monocolonies were seen in the lungs of patients who underwent transplantation, thesewere not the same microbes seen in the stomach; the microbial overlap between lung and gastric sam-ples within patients was low, and data indicated high individual variation between lung transplantrecipients. Other contributors to reduced alpha diversity included antibiotics in combination with pro-ton pump inhibitors, especially in gastric and oropharyngeal samples.CONCLUSIONS:Lung transplant recipients have reduced microbial diversity in gastric fluid (GF) andoropharynx compared with patients who did not undergo lung transplantation. The decreased alphadiversity in GF may be associated with dysmotility.J Heart Lung Transplant 000;000:1−10ÓPublished by Elsevier Inc. on behalf of International Society for Heart and Lung Transplantation.

Although the locations of promoters and enhancers have been identified in several cell types, we still have limited information on their connectivity. We developed HiCap, which combines a 4-cutter restriction enzyme Hi-C with sequence capture of promoter regions. Applying the method to mouse embryonic stem cells, we identified promoter-anchored interactions involving 15,905 promoters and 71,984 distal regions. The distal regions were enriched for enhancer marks and transcription, and had a mean fragment size of only 699 bp - close to single-enhancer resolution. High-resolution maps of promoter-anchored interactions with HiCap will be important for detailed characterizations of chromatin interaction landscapes.