NGI offers spatial transcriptomics through the 10X Genomics Visium method, a technique recently nominated as Method of the Year by Nature Methods. The technology combines histology with unbiased transcriptomics in a spatial context.
The Illumina NovaSeq 6000 system is the largest of the Illumina sequencing instruments, able to run two flow cells independently of each other and generate massive sequencing depth at competitive prices.
NGI is still up and running during the Covid-19 pandemic, but we are experiencing some limitations in terms of personnel and key reagents. Each NGI node is following its respective host university recommendations and will continue operation until further notice.
Would you like to work in a field with rapid development in technologies and applications? We are now recruiting a new member to join our team of project coordinators at NGI Uppsala (the SNP&SEQ Technology platform)
Tissue-specific transcriptional imprinting and heterogeneity in human innate lymphoid cells revealed by full-length single-cell RNA-sequencing
L Mazzurana, P Czarnewski, V Jonsson, L Wigge, M Ringnér, TC Williams, A Ravindran, ÅK Björklund, J Säfholm, G Nilsson, SE Dahlén, AC Orre, M Al-Ameri, C Höög, C Hedin, S Szczegielniak, S Almer, J Mjösberg
Cell Res, 31 (5) 1001-0602 (2021)
The impact of the microenvironment on innate lymphoid cell (ILC)-mediated immunity in humans remains largely unknown. Here we used full-length Smart-seq2 single-cell RNA-sequencing to unravel tissue-specific transcriptional profiles and heterogeneity of CD127 + ILCs across four human tissues. Correlation analysis identified gene modules characterizing the migratory properties of tonsil and blood ILCs, and signatures of tissue-residency, activation and modified metabolism in colon and lung ILCs. Trajectory analysis revealed potential differentiation pathways from circulating and tissue-resident naïve ILCs to a spectrum of mature ILC subsets. In the lung we identified both CRTH2+ and CRTH2- ILC2 with lung-specific signatures, which could be recapitulated by alarmin-exposure of circulating ILC2. Finally, we describe unique TCR-V(D)J-rearrangement patterns of blood ILC1-like cells, revealing a subset of potentially immature ILCs with TCR-δ rearrangement. Our study provides a useful resource for in-depth understanding of ILC-mediated immunity in humans, with implications for disease.
Comparative Fungal Community Analyses Using Metatranscriptomics and Internal Transcribed Spacer Amplicon Sequencing from Norway Spruce
AN Schneider, J Sundh, G Sundström, K Richau, N Delhomme, M Grabherr, V Hurry, NR Street
mSystems, 6 (1) 2379-5077 (2021)
The health, growth, and fitness of boreal forest trees are impacted and improved by their associated microbiomes. Microbial gene expression and functional activity can be assayed with RNA sequencing (RNA-Seq) data from host samples. In contrast, phylogenetic marker gene amplicon sequencing data are used to assess taxonomic composition and community structure of the microbiome. Few studies have considered how much of this structural and taxonomic information is included in transcriptomic data from matched samples. Here, we described fungal communities using both host-derived RNA-Seq and fungal ITS1 DNA amplicon sequencing to compare the outcomes between the methods. We used a panel of root and needle samples from the coniferous tree species Picea abies (Norway spruce) growing in untreated (nutrient-deficient) and nutrient-enriched plots at the Flakaliden forest research site in boreal northern Sweden. We show that the relationship between samples and alpha and beta diversity indicated by the fungal transcriptome is in agreement with that generated by the ITS data, while also identifying a lack of taxonomic overlap due to limitations imposed by current database coverage. Furthermore, we demonstrate how metatranscriptomics data additionally provide biologically informative functional insights. At the community level, there were changes in starch and sucrose metabolism, biosynthesis of amino acids, and pentose and glucuronate interconversions, while processing of organic macromolecules, including aromatic and heterocyclic compounds, was enriched in transcripts assigned to the genus CortinariusIMPORTANCE A deeper understanding of microbial communities associated with plants is revealing their importance for plant health and productivity. RNA extracted from plant field samples represents the host and other organisms present. Typically, gene expression studies focus on the plant component or, in a limited number of studies, expression in one or more associated organisms. However, metatranscriptomic data are rarely used for taxonomic profiling, which is currently performed using amplicon approaches. We created an assembly-based, reproducible, and hardware-agnostic workflow to taxonomically and functionally annotate fungal RNA-Seq data obtained from Norway spruce roots, which we compared to matching ITS amplicon sequencing data. While we identified some limitations and caveats, we show that functional, taxonomic, and compositional insights can all be obtained from RNA-Seq data. These findings highlight the potential of metatranscriptomics to advance our understanding of interaction, response, and effect between host plants and their associated microbial communities.
Immune cells lacking Y chromosome show dysregulation of autosomal gene expression.
JP Dumanski, J Halvardson, H Davies, E Rychlicka-Buniowska, J Mattisson, BT Moghadam, N Nagy, K Węglarczyk, K Bukowska-Strakova, M Danielsson, P Olszewski, A Piotrowski, E Oerton, A Ambicka, M Przewoźnik, Ł Bełch, T Grodzicki, PL Chłosta, S Imreh, V Giedraitis, L Kilander, J Nordlund, A Ameur, U Gyllensten, Å Johansson, A Józkowicz, M Siedlar, A Klich-Rączka, J Jaszczyński, S Enroth, J Baran, M Ingelsson, JRB Perry, J Ryś, LA Forsberg
NGI CollaborationCell. Mol. Life Sci., 78 (8) 1420-9071 (2021)
Epidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer's disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a "genetic wasteland", and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.
Candidate regulators and target genes of drought stress in needles and roots of Norway spruce.
JC Haas, A Vergara, AR Serrano, S Mishra, V Hurry, NR Street
Tree Physiol, 1758-4469 (2021)
Drought stress impacts seedling establishment, survival and whole-plant productivity. Molecular responses to drought stress have been most extensively studied in herbaceous species, mostly considering only aboveground tissues. Coniferous tree species dominate boreal forests, which are predicted to be exposed to more frequent and acute drought as a result of ongoing climate change. The associated impact at all stages of the forest tree life cycle is expected to have large-scale ecological and economic impacts. However, the molecular response to drought has not been comprehensively profiled for coniferous species. We assayed the physiological and transcriptional response of Picea abies (L.) H. Karst seedling needles and roots after exposure to mild and severe drought. Shoots and needles showed extensive reversible plasticity for physiological measures indicative of drought response mechanisms, including changes in stomatal conductance (gs), shoot water potential and ABA (abscisic acid). In both tissues the most commonly observed expression profiles in response to drought were highly correlated with ABA levels. Still, root and needle transcriptional responses contrasted, with extensive root-specific downregulation of growth. Comparison between previously characterized A. thaliana drought-response genes and P. abies revealed both conservation and divergence of transcriptional response to drought. In P. abies, transcription factors belonging to the bZIP AREB/ABF (ABA Response Element Binding/ABRE Binding Factors) ABA-dependent pathway had a more limited role. These results highlight the importance of profiling both above- and below-ground tissues and provide a comprehensive framework to advance understanding of the drought response of P. abies. The results demonstrate that short term, severe drought induces severe physiological responses coupled to extensive transcriptome modulation and highlight the susceptibility of Norway spruce seedlings to such drought events.
Long-read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome-gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic-based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18-chr8 integrations at base-pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.
Whole-genome analyses provide no evidence for dog introgression in Fennoscandian wolf populations.
L Smeds, J Aspi, J Berglund, I Kojola, K Tirronen, H Ellegren
Evol Appl, 14 (3) 1752-4571 (2021)
Hybridization and admixture can threaten the genetic integrity of populations and be of particular concern to endangered species. Hybridization between grey wolves and dogs has been documented in many wolf populations worldwide and is a prominent example of human-mediated hybridization between a domesticated species and its wild relative. We analysed whole-genome sequences from >200 wolves and >100 dogs to study admixture in Fennoscandian wolf populations. A principal component analysis of genetic variation and admixture showed that wolves and dogs were well-separated, without evidence for introgression. Analyses of local ancestry revealed that wolves had <1% mixed ancestry, levels comparable to the degree of mixed ancestry in many dogs, and likely not resulting from recent wolf-dog hybridization. We also show that the founders of the Scandinavian wolf population were genetically inseparable from Finnish and Russian Karelian wolves, pointing at the geographical origin of contemporary Scandinavian wolves. Moreover, we found Scandinavian-born animals among wolves sampled in Finland, demonstrating bidirectional gene flow between the Scandinavian Peninsula and eastern countries. The low incidence of admixture between wolves and dogs in Fennoscandia may be explained by the fact that feral dogs are rare in this part of Europe and that careful monitoring and management act to remove hybrids before they backcross into wolf populations.
Long-read single molecule sequencing is increasingly used in human genomics research, as it allows to accurately detect large-scale DNA rearrangements such as structural variations (SVs) at high resolution. However, few studies have evaluated the performance of different single molecule sequencing platforms for SV detection in human samples. Here we performed Oxford Nanopore Technologies (ONT) whole-genome sequencing of two Swedish human samples (average 32× coverage) and compared the results to previously generated Pacific Biosciences (PacBio) data for the same individuals (average 66× coverage). Our analysis inferred an average of 17k and 23k SVs from the ONT and PacBio data, respectively, with a majority of them overlapping with an available multi-platform SV dataset. When comparing the SV calls in the two Swedish individuals, we find a higher concordance between ONT and PacBio SVs detected in the same individual as compared to SVs detected by the same technology in different individuals. Downsampling of PacBio reads, performed to obtain similar coverage levels for all datasets, resulted in 17k SVs per individual and improved overlap with the ONT SVs. Our results suggest that ONT and PacBio have a similar performance for SV detection in human whole genome sequencing data, and that both technologies are feasible for population-scale studies.
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