Galaxy is a mature, browser accessible workbench for scientific computing. It enables scientists to share, analyze and visualize their own data, with minimal technical impediments. A thriving global community continues to use, maintain and contribute to the project, with support from multiple national infrastructure providers that enable freely accessible analysis and training services. The Galaxy Training Network supports free, self-directed, virtual training with >230 integrated tutorials. Project engagement metrics have continued to grow over the last 2 years, including source code contributions, publications, software packages wrapped as tools, registered users and their daily analysis jobs, and new independent specialized servers. Key Galaxy technical developments include an improved user interface for launching large-scale analyses with many files, interactive tools for exploratory data analysis, and a complete suite of machine learning tools. Important scientific developments enabled by Galaxy include Vertebrate Genome Project (VGP) assembly workflows and global SARS-CoV-2 collaborations.

Illumina and nanopore sequencing technologies are powerful tools that can be used to determine the bacterial composition of complex microbial communities. In this study, we compared nasal microbiota results at genus level using both Illumina and nanopore 16S rRNA gene sequencing. We also monitored the progression of nanopore sequencing in the accurate identification of species, using pure, single species cultures, and evaluated the performance of the nanopore EPI2ME 16S data analysis pipeline. Fifty-nine nasal swabs were sequenced using Illumina MiSeq and Oxford Nanopore 16S rRNA gene sequencing technologies. In addition, five pure cultures of relevant bacterial species were sequenced with the nanopore sequencing technology. The Illumina MiSeq sequence data were processed using bioinformatics modules present in the Mothur software package. Albacore and Guppy base calling, a workflow in nanopore EPI2ME (Oxford Nanopore Technologies—ONT, Oxford, UK) and an in-house developed bioinformatics script were used to analyze the nanopore data. At genus level, similar bacterial diversity profiles were found, and five main and established genera were identified by both platforms. However, probably due to mismatching of the nanopore sequence primers, the nanopore sequencing platform identified Corynebacterium in much lower abundance compared to Illumina sequencing. Further, when using default settings in the EPI2ME workflow, almost all sequence reads that seem to belong to the bacterial genus Dolosigranulum and a considerable part to the genus Haemophilus were only identified at family level. Nanopore sequencing of single species cultures demonstrated at least 88% accurate identification of the species at genus and species level for 4/5 strains tested, including improvements in accurate sequence read identification when the basecaller Guppy and Albacore, and when flowcell versions R9.4 (Oxford Nanopore Technologies—ONT, Oxford, UK) and R9.2 (Oxford Nanopore Technologies—ONT, Oxford, UK) were compared. In conclusion, the current study shows that the nanopore sequencing platform is comparable with the Illumina platform in detection bacterial genera of the nasal microbiota, but the nanopore platform does have problems in detecting bacteria within the genus Corynebacterium. Although advances are being made, thorough validation of the nanopore platform is still recommendable.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor prognosis even after curative resection. Responses to immunotherapy are rare and related to inadequate T-cell priming. We previously demonstrated the potency of allogeneic lysate-dendritic cell (DC) vaccination in a preclinical model. Here we translate this concept to patients. In this phase I study, patients with resected PDAC were included when they demonstrated no radiologic signs of recurrence after standard-of-care treatment. Allogeneic tumour lysate-loaded autologous monocyte-derived DCs were injected at weeks 0, 2, 4 and at months 3 and 6. Objectives are feasibility, safety and immunogenicity of allogeneic tumour-DCs. The presence of tumour antigens shared between the vaccine and patient tumours was investigated. Immunological analyses were performed on peripheral blood, skin and tumour. Ten patients were included. DC production and administration were successful. All patients experienced a grade 1 injection-site and infusion-related reaction. Two patients experienced a grade 2 fever and 1 patient experienced a grade 3 dyspnoea. No vaccine-related serious adverse events were observed. Shared tumour antigens were found between the vaccine and patient tumours. All evaluated patients displayed a vaccine-induced response indicated by increased frequencies of Ki67+ and activated PD-1+ circulating T-cells. In addition, treatment-induced T-cell reactivity to autologous tumour of study patients was detected. Seven out of ten patients have not experienced disease recurrence or progression at a median follow-up of 25 months (15-32 months). Allogeneic tumour lysate-DC treatment is feasible, safe and induces immune reactivity to PDAC expressed antigens.

Aims: Lung tissue from COVID‐19 patients shares similar histomorphological features with chronic lung allograft disease, also suggesting activation of autoimmune‐related pathways in COVID‐19. To more clearly understand the underlying spectrum of pathophysiology in COVID‐19 pneumonia, we analysed mRNA expression of autoimmune‐related genes in post‐mortem lung tissue from COVID‐19 patients.Methods and results: Formalin‐fixed, paraffin‐embedded lung tissue samples of 18 COVID‐19 patients and eight influenza patients were used for targeted gene expression profiling using NanoString technology. Multiplex immunofluorescence for tryptase and chymase was applied for validation. Genes related to mast cells were significantly increased in COVID‐19. This finding was strengthened by multiplex immunofluorescence also showing a significant increase of tryptase‐ and chymase‐positive cells in COVID‐19. Furthermore, receptors for advanced glycation end‐products (RAGE) and pro‐platelet basic protein (PPBP) were up‐regulated in COVID‐19 compared to influenza. Genes associated with Type I interferon signalling showed a significant correlation to detected SARS‐CoV2 pathway‐related genes. The comparison of lung tissue samples from both groups based on the presence of histomorphological features indicative of acute respiratory distress syndrome did not result in finding any specific gene or pathways.Conclusion: Two separate means of measuring show a significant increase of mast cells in SARS‐CoV‐2‐infected lung tissue compared to influenza. Additionally, several genes involved in fibrosis and thrombosis, among which are RAGE and PPBP, are up‐regulated in COVID‐19. As mast cells are able to induce thrombosis and fibrosis, they may play an important role in the pathogenesis of COVID‐19.

Monitoring changes in the immune profile in blood samples can help identifying changes in tumor biology and therapy responsiveness over time. Immune-related gene expression profiles offer a highly reproducible method to monitor changes of the immune system. However, measuring gene expression profiles in whole blood samples can be complicated because of the high protein and enzyme abundancy that affect the stability and quality of the RNA. Peripheral blood mononuclear cells (PBMCs) are one the most commonly used source for immune cell RNA extraction, though, this method does not reflect all components of the peripheral blood. The aim of this study was to determine the differences in immune-related gene expression between RNA isolated from stabilized whole blood and RNA isolated from PBMCs. Whole blood samples from 12 pancreatic cancer patients were collected before and after chemotherapy (n = 24). Blood samples were collected in both EDTA tubes, and Tempus tubes containing an RNA stabilizer (total n = 48). PBMCs were isolated from EDTA samples using Ficoll and were snap frozen. Subsequently, immune-related gene expression was profiled using the PanCancer Immune Profiling Panel of NanoString technology. Gene expression profiles of PBMCs were compared to that of Tempus tubes using the Advanced Analysis module of nSolver software. Both types of samples provided good quality RNA and gene expression measurements. However, RNA isolated from Tempus tubes resulted in significantly higher gene counts than PBMCs; 107/730 genes were exclusively detected in Tempus samples, while under the detection limit in PBMCs. In addition, 192/730 genes showed significantly higher gene counts in Tempus samples, 157/730 genes showed higher gene counts in PBMCs. Thus, RNA isolated from whole blood stabilizing blood tubes, such as Tempus tubes, enable higher gene counts and more comprehensive measurements of gene expression profiles compared to RNA isolated from PBMCs.

Cervical cancer is one of the most common cancers in women worldwide. Patients diagnosed with early-stage cervical cancer have a good prognosis, however, 10-20% suffer from local or distant recurrent disease after primary treatment. Treatment options for recurrent cervical cancer are limited. Therefore, it is crucial to identify factors that can predict patients with an increased risk of recurrence to optimize treatment to prevent the recurrence of cervical cancer. We aimed to identify biomarkers in early-stage primary cervical cancer which recurred after surgery. Formalin-Fixed, Paraffin-Embedded surgical specimens of 34 patients with early-stage cervical cancer (FIGO 2009 stage 1B1) and 7 healthy controls were analyzed. Targeted gene expression profiling using the PanCancer IO 360 panel of NanoString Technology was performed. The findings were confirmed by performing immunohistochemistry stainings. Various genes, namely GLS, CD36, WNT5a, HRAS, DDB2, PIK3R2, and CDH2 were found to be differentially highly expressed in primary cervical cancer samples of patients who developed distant recurrence. In addition, The relative infiltration score of CD8+ T cells, CD80+CD86+ macrophages, CD163+MRC1+ macrophages, and FOXP3+IL2RA+ regulatory T cells were significantly higher in this group of samples. In contrast, no significant differences in gene expression and relative immune infiltration were found in samples of patients who developed local recurrence. The infiltration of CD8 and FOXP3 cells were validated by immunohistochemistry using all samples included in the study. We identified molecular alterations in primary cervical cancer samples from patients who developed recurrent disease. These findings can be utilized towards developing a molecular signature for the early detection of patients with a high risk to develop metastasis.