| 1. |
- Asp, Michaela, et al.
(författare)
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Spatial Isoform Profiling within Individual Tissue Sections
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Spatial Transcriptomics has been shown to be a persuasive RNA sequencingtechnology for analyzing cellular heterogeneity within tissue sections. Thetechnology efficiently captures and barcodes 3’ tags of all polyadenylatedtranscripts from a tissue section, and thus provides a powerful platform whenperforming quantitative spatial gene expression studies. However, the currentprotocol does not recover the full-length information of transcripts, andconsequently lack information regarding alternative splice variants. Here, weintroduce a novel protocol for spatial isoform profiling, using SpatialTranscriptomics barcoded arrays.
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| 2. |
- Berglund, Emelie, et al.
(författare)
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Automation of Spatial Transcriptomics library preparation to enable rapid and robust insights into spatial organization of tissues
- 2020
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Ingår i: BMC Genomics. - : Springer Nature. - 1471-2164. ; 21:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Interest in studying the spatial distribution of gene expression in tissues is rapidly increasing. Spatial Transcriptomics is a novel sequencing-based technology that generates high-throughput information on the distribution, heterogeneity and co-expression of cells in tissues. Unfortunately, manual preparation of high-quality sequencing libraries is time-consuming and subject to technical variability due to human error during manual pipetting, which results in sample swapping and the accidental introduction of batch effects. All these factors complicate the production and interpretation of biological datasets.Results: We have integrated an Agilent Bravo Automated Liquid Handling Platform into the Spatial Transcriptomics workflow. Compared to the previously reported Magnatrix 8000+ automated protocol, this approach increases the number of samples processed per run, reduces sample preparation time by 35%, and minimizes batch effects between samples. The new approach is also shown to be highly accurate and almost completely free from technical variability between prepared samples.Conclusions: The new automated Spatial Transcriptomics protocol using the Agilent Bravo Automated Liquid Handling Platform rapidly generates high-quality Spatial Transcriptomics libraries. Given the wide use of the Agilent Bravo Automated Liquid Handling Platform in research laboratories and facilities, this will allow many researchers to quickly create robust Spatial Transcriptomics libraries.
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| 3. |
- Engström, Karin, et al.
(författare)
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Transcriptomics and methylomics of CD4-positive T cells in arsenic-exposed women
- 2017
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 91:5, s. 2067-2078
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Tidskriftsartikel (refereegranskat)abstract
- Arsenic, a carcinogen with immunotoxic effects, is a common contaminant of drinking water and certain food worldwide. We hypothesized that chronic arsenic exposure alters gene expression, potentially by altering DNA methylation of genes encoding central components of the immune system. We therefore analyzed the transcriptomes (by RNA sequencing) and methylomes (by target-enrichment next-generation sequencing) of primary CD4-positive T cells from matched groups of four women each in the Argentinean Andes, with fivefold differences in urinary arsenic concentrations (median concentrations of urinary arsenic in the lower- and high-arsenic groups: 65 and 276 mu g/l, respectively). Arsenic exposure was associated with genome-wide alterations of gene expression; principal component analysis indicated that the exposure explained 53% of the variance in gene expression among the top variable genes and 19% of 28,351 genes were differentially expressed (false discovery rate < 0.05) between the exposure groups. Key genes regulating the immune system, such as tumor necrosis factor alpha and interferon gamma, as well as genes related to the NF-kappa-beta complex, were significantly downregulated in the high-arsenic group. Arsenic exposure was associated with genome-wide DNA methylation; the high-arsenic group had 3% points higher genome-wide full methylation (> 80% methylation) than the lower-arsenic group. Differentially methylated regions that were hyper-methylated in the high-arsenic group showed enrichment for immune-related gene ontologies that constitute the basic functions of CD4-positive T cells, such as isotype switching and lymphocyte activation and differentiation. In conclusion, chronic arsenic exposure from drinking water was related to changes in the transcriptome and methylome of CD4-positive T cells, both genome wide and in specific genes, supporting the hypothesis that arsenic causes immunotoxicity by interfering with gene expression and regulation.
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| 4. |
- Grimm, Sebastian, et al.
(författare)
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Single domain affinity proteins for the detection of the genome organizer protein SATB1
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Affibody molecules and VHH antibody fragments are two classes of affinity proteins, both characterized by a small size and a single subunit domain structure. Here, we report the selection and characterization of affinity proteins from both classes against the special AT rich sequence binding protein SATB1, a suggested marker protein for aggressive and metastasizing breast cancer. The selected VHH antibody fragments originate from a nonimmune phage display library, while affibody molecules were selected from a library constructed in vitro and displayed on ribosomes. It was observed that selected molecules recognizing one of three conserved DNA-binding domains of SATB1, also recognized its close homologue SATB2 while several of the selected molecules from both classes binding to other regions selectively recognized SATB1. Two of these SATB1 selective molecules, VHH clone 2D2 and affibody molecule clone ZSATB1:2, performed well in differentimmunotechnology applications including ELISA, WB, IF and pull-out experiments and gave a selective staining of endogenous SATB1 in Jurkat T cells. These molecules may thus become useful tools, either alone or in combination, for the selective detection of SATB1 in breast tumor specimens. Due to their small size in comparison to immunoglobulins, such single domain binding proteins may be suitable for high resolution microscopy techniques such as Stimulated Emission Depletion (STED) microscopy, where the resolution may get constrained by the size of the affinity reagent.
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| 5. |
- Jemt, Anders, 1985-, et al.
(författare)
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Evaluation of methods for whole genome and transcriptome sequencing from nanograms of FFPE samples
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The most widely used method for the preservation of clinical tissue specimens is formalin fixation and paraffin embedding (FFPE). Simultaneous analysis of RNA and DNA from samples preserved using this method have long proved problematic, primarily due to lack of material. Here, we describe an attempt to build a complete analysis package for RNA and DNA extracted from single tissue sections. The workflow includes quality control of the extracted material, library preparation and data analysis. We extract DNA with varying integrity from FFPE sections and subject them to whole genome sequencing using two library preparation methods, Illumina TruSeq Nano using the Illumina NeoPrep and Rubicon Genomics ThruPlex. We are able to obtain some usable data, albeit with high duplication rates, demonstrating both the possibilities and challenges of sequencing damaged DNA. Two different approaches to transcriptome sequencing are assessed, the TruSeq RNA Access library preparation kit from Illumina and the SMARTer Stranded Total RNA-Seq Kit - Pico Input from Clonetech. The sequence capture approach of the TruSeq kit is shown to be more robust to low integrity RNA compared to the SMARTer kit. However, the SMARTer kit needs much less starting material and is able to yield data about all transcripts, not just protein coding mRNA.
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| 6. |
- Lundin, Sverker, et al.
(författare)
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Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
- 2013
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3, s. 1186-
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Tidskriftsartikel (refereegranskat)abstract
- Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.
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| 7. |
- Lutgen, Dave, et al.
(författare)
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Linked-read sequencing enables haplotype-resolved resequencing at population scale
- 2020
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Ingår i: Molecular Ecology Resources. - : WILEY. - 1755-098X .- 1755-0998. ; 20:5, s. 1311-1322
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Tidskriftsartikel (refereegranskat)abstract
- The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences - including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps - are still limited by the lack of high-quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype-resolved genome resequencing at population scale, we investigated properties of linked-read sequencing data of songbirds of the genusOenantheacross a range of sequencing depths. Our results based on the comparison of downsampled (25x, 20x, 15x, 10x, 7x, and 5x) with high-coverage data (46-68x) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15x coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25-4.6 Mb (N50) and 0.27-0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15x coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25x coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher-quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase-sized genomes like birds, linked-read sequencing at moderate depth opens an affordable avenue towards haplotype-resolved genome resequencing at population scale.
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| 8. |
- Mueller, Jakob C., et al.
(författare)
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Local selection signals in the genome of blue tits emphasize regulatory and neuronal evolution
- 2022
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Ingår i: Molecular Ecology. - : Wiley. - 0962-1083 .- 1365-294X. ; 31:5, s. 1504-1514
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the genomic landscape of adaptation is central to understanding microevolution in wild populations. Genomic targets of selection and the underlying genomic mechanisms of adaptation can be elucidated by genome-wide scans for past selective sweeps or by scans for direct fitness associations. We sequenced and assembled 150 haplotypes of 75 blue tits (Cyanistes caeruleus) of a single Central European population by a linked-read technology. We used these genome data in combination with coalescent simulations (i) to estimate an historical effective population size of ~250,000, which recently declined to ~10,000, and (ii) to identify genome-wide distributed selective sweeps of beneficial variants probably originating from standing genetic variation (soft sweeps). The genes linked to these soft sweeps, but also those linked to hard sweeps based on new beneficial mutants, showed a significant enrichment for functions associated with gene expression and transcription regulation. This emphasizes the importance of regulatory evolution in the population’s adaptive history. Soft sweeps were further enriched for genes related to axon and synapse development, indicating the significance of neuronal connectivity changes in the brain potentially linked to behavioural adaptations. A previous scan of heterozygosity–fitness correlations revealed a consistent negative effect on arrival date at the breeding site for a single microsatellite in the MDGA2 gene. Here, we used the haplotype structure around this microsatellite to explain the effect as a local and direct outbreeding effect of a gene involved in synapse development.
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| 9. |
- Schweizer, Manuel, et al.
(författare)
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Genome-wide evidence supports mitochondrial relationships and pervasive parallel phenotypic evolution in open-habitat chats
- 2019
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Ingår i: Molecular Phylogenetics and Evolution. - : Elsevier BV. - 1055-7903 .- 1095-9513. ; 139
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Tidskriftsartikel (refereegranskat)abstract
- In wheatears and related species ('open-habitat chats'), molecular phylogenetics has led to a comprehensively revised understanding of species relationships and species diversity. Phylogenetic analyses have suggested that, in many cases, phenotypic similarities do not reflect species' relationships, revealing traditionally defined genera as non-monophyletic. This led to the suggestion of pervasive parallel evolution of open-habitat chats' plumage coloration and ecological phenotypes. However, to date, the molecular evidence for the phylogenetic relationships among open-habitat chats is mainly limited to mitochondrial DNA. Here, we assessed whether the mitochondrial relationships are supported by genome-wide data. To this end, we reconstructed the species tree among 14 open-habitat chat taxa using multi-species coalescent analyses based on similar to 1'300 SNPs. Our results confirm previous ones based chiefly on mitochondrial DNA; notably the paraphyly of the Oenanthe lugens complex and the clustering of individual species formerly placed in the genera Cercomela and Myrmecocichla within Oenanthe. Since several variable morphological and ecological characteristics occur in multiple places across the open-habitat chat phylogeny, our study consolidates the evidence for pervasive parallel evolution in the plumage coloration and ecology of open-habitat chats.
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