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  • 101. Falkenius, J
    et al.
    Lindberg, J
    Johansson, H.
    Frostvik-Stolt, M.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Hansson, J.
    Egyhazi, S.
    A gene expression profile associated with clinical outcome in stage III melanoma2011In: Journal of Clinical Oncology, ISSN 0732-183X, E-ISSN 1527-7755, Vol. 29, no 15Article in journal (Other academic)
  • 102. Falkenius, Johan
    et al.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Johansson, Hemming
    Tuominen, Rainer
    Frostvik-Stolt, Marianne
    Hansson, Johan
    Brage, Suzanne Egyhazi
    High expression of glycolytic and pigment proteins is associated with worse clinical outcome in stage III melanoma2013In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, Vol. 23, no 6, p. 452-460Article in journal (Refereed)
    Abstract [en]

    There are insufficient numbers of prognostic factors available for prediction of clinical outcome in patients with stage III malignant cutaneous melanoma, even when known adverse pathological risk factors, such as macrometastasis, number of lymph node metastases, and ulceration are taken into consideration. The aim of this study was therefore to identify additional prognostic factors to better predict patients with a high risk of relapse, thus enabling us to better determine the need for adjuvant treatment in stage III disease. An RNA oligonucleotide microarray study was performed on first regional lymph node metastases in 42 patients with stage III melanoma: 23 patients with short-term survival (13 months) and 19 with long-term survival (60 months), to identify genes associated with clinical outcome. Candidate genes were validated by real-time PCR and immunohistochemical analysis. Several gene ontology (GO) categories were highly significantly differentially expressed including glycolysis (GO: 0006096; P<0.001) and the pigment biosynthetic process (GO: 0046148; P<0.001), in which overexpression was associated with short-disease-specific survival. Three overexpressed glycolytic genes, GAPDHS, GAPDH, and PKM2, and two pigment-related genes, TYRP1 and OCA2, were selected for validation. A significant difference in GAPDHS protein expression between short- and long-term survivors (P=0.021) and a trend for PKM2 (P=0.093) was observed in univariate analysis. Positive expression of at least two of four proteins (GAPDHS, GAPDH, PKM2, TYRP1) in immunohistochemical analysis was found to be an independent adverse prognostic factor for disease-specific survival (P=0.011). Our results indicate that this prognostic panel in combination with established risk factors may contribute to an improved prediction of patients with a high risk of relapse.

  • 103. Farnelid, Hanna
    et al.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bertilsson, Stefan
    Abu Al-Soud, Waleed
    Hansen, Lars H.
    Sørensen, Søren
    Steward, Grieg F.
    Hagstöom, Åke
    Riemann, Lasse
    Nitrogenase Gene Amplicons from Global Marine Surface Waters Are Dominated by Genes of Non-Cyanobacteria2011In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 4, p. e19223-Article in journal (Refereed)
    Abstract [en]

    Cyanobacteria are thought to be the main N-2-fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations worldwide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of non-cyanobacterial diazotrophs to the global N-2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.

  • 104. Farnelid, Hanna
    et al.
    Bentzon-Tilia, Mikkel
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bertilsson, Stefan
    Jost, Guenter
    Labrenz, Matthias
    Juergens, Klaus
    Riemann, Lasse
    Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea2013In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 7, no 7, p. 1413-1423Article in journal (Refereed)
    Abstract [en]

    The Baltic Sea receives large nitrogen inputs by diazotrophic (N-2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N-2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N-2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of >10 and <10 mu m size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 x 10(7) nifH copies l(-1). Abundant nifH transcripts (up to 3.2 x 10(4) transcripts l(-1)) within nifH cluster III and co-occurring N-2 fixation (0.44 +/- 0.26 nmol l(-1) day(-1)) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N-2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N-2 fixation could also be of importance in other suboxic regions of the world's oceans.

  • 105. Fletcher, E.
    et al.
    Feizi, A.
    Bisschops, M. M. M.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Khoomrung, S.
    Siewers, V.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Evolutionary engineering reveals divergent paths when yeast is adapted to different acidic environments2017In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 39, p. 19-28Article in journal (Refereed)
    Abstract [en]

    Tolerance of yeast to acid stress is important for many industrial processes including organic acid production. Therefore, elucidating the molecular basis of long term adaptation to acidic environments will be beneficial for engineering production strains to thrive under such harsh conditions. Previous studies using gene expression analysis have suggested that both organic and inorganic acids display similar responses during short term exposure to acidic conditions. However, biological mechanisms that will lead to long term adaptation of yeast to acidic conditions remains unknown and whether these mechanisms will be similar for tolerance to both organic and inorganic acids is yet to be explored. We therefore evolved Saccharomyces cerevisiae to acquire tolerance to HCl (inorganic acid) and to 0.3 M L-lactic acid (organic acid) at pH 2.8 and then isolated several low pH tolerant strains. Whole genome sequencing and RNA-seq analysis of the evolved strains revealed different sets of genome alterations suggesting a divergence in adaptation to these two acids. An altered sterol composition and impaired iron uptake contributed to HCl tolerance whereas the formation of a multicellular morphology and rapid lactate degradation was crucial for tolerance to high concentrations of lactic acid. Our findings highlight the contribution of both the selection pressure and nature of the acid as a driver for directing the evolutionary path towards tolerance to low pH. The choice of carbon source was also an important factor in the evolutionary process since cells evolved on two different carbon sources (raffinose and glucose) generated a different set of mutations in response to the presence of lactic acid. Therefore, different strategies are required for a rational design of low pH tolerant strains depending on the acid of interest.

  • 106. Forsell, M. N. E.
    et al.
    Kvastad, Linda
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sedimbi, S. K.
    Andersson, J.
    Karlsson, M. C. I.
    Regulation of subunit-specific germinal center B cell responses to the HIV-1 envelope glycoproteins by antibody-mediated feedback2017In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, no JUN, article id 738Article in journal (Refereed)
    Abstract [en]

    The regulation of germinal center (GC) B cell responses to single epitopes is well investigated. How monoclonal B cells are regulated within the polyclonal B cell response to protein antigens is less so. Here, we investigate the primary GC B cell response after injection of mice with HIV-1 envelope glycoproteins. We demonstrate that single GCs are seeded by a diverse number of B cell clones shortly after a single immunization and that the presence of Env-specific antibodies can inhibit the development of early GC B cells. Importantly, the suppression was dependent on the GC B cells and the infused antibodies to target the same subunit of the injected HIV-1 envelope glycoproteins. An affinity-dependent antibody feedback has previously been shown to regulate GC B cell development. Here, we propose that this antibody-based feedback acts on GC B cells only if they target the same or overlapping epitopes. This study provides important basic information of GC B cell regulation, and for future vaccine designs with aim to elicit neutralizing antibodies against HIV-1.

  • 107. Fransson, Martin N.
    et al.
    Brugård, Jan
    Aronsson, Peter
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Semi-physiologically based pharmacokinetic modeling of paclitaxel metabolism and in silico-based study of the dynamic sensitivities in pathway kinetics2012In: European Journal of Pharmaceutical Sciences, ISSN 0928-0987, E-ISSN 1879-0720, Vol. 47, no 4, p. 759-767Article in journal (Refereed)
    Abstract [en]

    Purpose: To build a semi-physiologically based pharmacokinetic model describing the uptake, metabolism and efflux of paclitaxel and its metabolites and investigate the effect of hypothetical genetic polymorphisms causing reduced uptake, metabolism or efflux in the pathway by model simulation and sensitivity analysis. Methods: A previously described intracellular pharmacokinetic model was used as a starting point for model development. Kinetics for metabolism, transport, binding and systemic and output compartments were added to mimic a physiological model with hepatic elimination. Model parameters were calibrated using constraints postulated as ratios of concentrations and amounts of metabolites and drug in the systemic plasma and output compartments. The sensitivity in kinetic parameters was tested using dynamic sensitivity analysis. Results: Predicted plasma concentrations of drug and metabolites were in the range of what has been observed in clinical studies. Given the final model, plasma concentrations of paclitaxel seems to be relatively little affected by changes in metabolism or transport, while its main metabolite may be largely affected even by small changes. If metabolites prove to be clinically relevant, genetic polymorphisms may play an important role for individualizing paclitaxel treatment.

  • 108. Friboulet, Luc
    et al.
    Barrios-Gonzales, Daniel
    Commo, Frederic
    Olaussen, Ken Andre
    Vagner, Stephan
    Adam, Julien
    Goubar, Aicha
    Dorvault, Nicolas
    Lazar, Vladimir
    Job, Bastien
    Besse, Benjamin
    Validire, Pierre
    Girard, Philippe
    Lacroix, Ludovic
    Hasmats, Johanna
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Dufour, Fabienne
    Andre, Fabrice
    Soria, Jean-Charles
    Molecular Characteristics of ERCC1-Negative versus ERCC1-Positive Tumors in Resected NSCLC2011In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 17, no 17, p. 5562-5572Article in journal (Refereed)
    Abstract [en]

    Purpose: Excision repair cross-complementation group 1 (ERCC1) is a protein involved in repair of DNA platinum adducts and stalled DNA replication forks. We and others have previously shown the influence of ERCC1 expression upon survival rates and benefit of cisplatin-based chemotherapy in patients with resected non-small-cell lung cancer (NSCLC). However, little is known about the molecular characteristics of ERCC1-positive and ERCC1-negative tumors. Experimental Design: We took advantage of a cohort of 91 patients with resected NSCLC, for which we had matched frozen and paraffin-embedded samples to explore the comparative molecular portraits of ERCC1-positive and ERCC1-negative tumors of NSCLC. We carried out a global molecular analysis including assessment of ERCC1 expression levels by using both immunohistochemistry (IHC) and quantitative reverse transcriptase PCR (qRT-PCR), genomic instability, global gene and miRNA expression, and sequencing of selected key genes involved in lung carcinogenesis. Results: ERCC1 protein and mRNA expression were significantly correlated. However, we observed several cases with clear discrepancies. We noted that ERCC1-negative tumors had a higher rate of genomic abnormalities versus ERCC1-positive tumors. ERCC1-positive tumors seemed to share a common DNA damage response (DDR) phenotype with the overexpression of seven genes linked to DDR. The miRNA expression analysis identified miR-375 as significantly underexpressed in ERCC1-positive tumors. Conclusions: Our data show inconsistencies in ERCC1 expression between IHC and qRT-PCR readouts. Furthermore, ERCC1 status is not linked to specific mutational patterns or frequencies. Finally, ERCC1negative tumors have a high rate of genomic aberrations that could consequently influence prognosis in patients with resected NSCLC. Clin Cancer Res; 17(17); 5562-72.

  • 109. Frings, O.
    et al.
    Alexeyenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Stockholm Bioinformatics Centre, Science for Life Laboratory, Solna, Sweden .
    Sonnhammer, E. L. L.
    MGclus: Network clustering employing shared neighbors2013In: Molecular BioSystems, ISSN 1742-206X, Vol. 9, no 7, p. 1670-1675Article in journal (Refereed)
    Abstract [en]

    Network analysis is an important tool for functional annotation of genes and proteins. A common approach to discern structure in a global network is to infer network clusters, or modules, and assume a functional coherence within each module, which may represent a complex or a pathway. It is however not trivial to define optimal modules. Although many methods have been proposed, it is unclear which methods perform best in general. It seems that most methods produce far from optimal results but in different ways. MGclus is a new algorithm designed to detect modules with a strongly interconnected neighborhood in large scale biological interaction networks. In our benchmarks we found MGclus to outperform other methods when applied to random graphs with varying degree of noise, and to perform equally or better when applied to biological protein interaction networks. MGclus is implemented in Java and utilizes the JGraphT graph library. It has an easy to use command-line interface and is available for download from http://sonnhammer.sbc.su.se/download/software/ MGclus/.

  • 110.
    Frings, Oliver
    et al.
    Stockholm Univ, Science for life laboratory.
    Mank, Judith E.
    Alexeyenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sonnhammer, Erik L. L.
    Network Analysis of Functional Genomics Data: Application to Avian Sex-Biased Gene Expression2012In: Scientific World Journal, ISSN 1537-744X, E-ISSN 1537-744X, p. 130491-Article in journal (Refereed)
    Abstract [en]

    Gene expression analysis is often used to investigate the molecular and functional underpinnings of a phenotype. However, differential expression of individual genes is limited in that it does not consider how the genes interact with each other in networks. To address this shortcoming we propose a number of network-based analyses that give additional functional insights into the studied process. These were applied to a dataset of sex-specific gene expression in the chicken gonad and brain at different developmental stages. We first constructed a global chicken interaction network. Combining the network with the expression data showed that most sex-biased genes tend to have lower network connectivity, that is, act within local network environments, although some interesting exceptions were found. Genes of the same sex bias were generally more strongly connected with each other than expected. We further studied the fates of duplicated sex-biased genes and found that there is a significant trend to keep the same pattern of sex bias after duplication. We also identified sex-biased modules in the network, which reveal pathways or complexes involved in sex-specific processes. Altogether, this work integrates evolutionary genomics with systems biology in a novel way, offering new insights into the modular nature of sex-biased genes.

  • 111. Gantner, S
    et al.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Alonso-Saez, L
    Bertilsson, S
    Novel primers for 16S rRNA-based archaeal community analyses in environmental samples2011In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 84, no 1, p. 12-18Article in journal (Refereed)
    Abstract [en]

    Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (<1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples.

  • 112. Ghaffari, Pouyan
    et al.
    Mardinoglu, Adil
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden; Technical University of Denmark, Denmark.
    Cancer Metabolism: A Modeling Perspective2015In: Frontiers in Physiology, ISSN 1664-042X, E-ISSN 1664-042X, Vol. 6, article id 382Article, review/survey (Refereed)
    Abstract [en]

    Tumor cells alter their metabolism to maintain unregulated cellular proliferation and survival, but this transformation leaves them reliant on constant supply of nutrients and energy. In addition to the widely studied dysregulated glucose metabolism to fuel tumor cell growth, accumulating evidences suggest that utilization of amino acids and lipids contributes significantly to cancer cell metabolism. Also recent progresses in our understanding of carcinogenesis have revealed that cancer is a complex disease and cannot be understood through simple investigation of genetic mutations of cancerous cells. Cancer cells present in complex tumor tissues communicate with the surrounding microenvironment and develop traits which promote their growth, survival, and metastasis. Decoding the full scope and targeting dysregulated metabolic pathways that support neoplastic transformations and their preservation requires both the advancement of experimental technologies for more comprehensive measurement of omics as well as the advancement of robust computational methods for accurate analysis of the generated data. Here, we review cancer-associated reprogramming of metabolism and highlight the capability of genome-scale metabolic modeling approaches in perceiving a system-level perspective of cancer metabolism and in detecting novel selective drug targets.

  • 113. Gharizadeh, B.
    et al.
    Oggionni, M.
    Zheng, B. Y.
    Akom, E.
    Pourmand, N.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wallin, K. L.
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry.
    Type-specific multiple sequencing primers - A novel strategy for reliable and rapid genotyping of human papilloma viruses by pyrosequencing technology2005In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 7, no 2, p. 198-205Article in journal (Refereed)
    Abstract [en]

    DNA sequencing is the gold standard method for accurate microbial and viral typing. However, DNA sequencing techniques have been facing limitations in typing of human papillomaviruses when the specimen harbors multiple genotypes and yields nonspecific amplification products, resulting in nonspecific and noninterpretable sequence data. To address these limitations we have developed a type-specific multiple sequencing primer DNA-sequencing method. This new strategy is suitable for sequencing and typing of samples harboring different genotypes (co-infections with multiple genotypes) and yielding nonspecific amplifications, thus eliminating the need for nested polymerase chain reaction (PCR), stringent PCR conditions, and cloning. The new approach has also proved useful for amplicons containing low PCR yield or subdominant types, avoiding reperforming of amplifications. We have applied the multiple sequencing primer method for genotyping of clinically relevant human papillomaviruses in a clinical test panel by using a combined pool of seven type-specific sequencing primers for HPV-6, -11, -16, -18, -31, -33, and -45. Furthermore, we introduced a sequence pattern recognition approach when there was a plurality of genotypes in the sample to facilitate typing of more than one target DNA in the sample. The multiple sequencing primer method has proved to be a multifaceted approach for typing of human papillomaviruses by DNA sequencing technologies.

  • 114.
    Giacomello, Stefania
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Stockholm University, Sweden.
    Salmén, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Terebieniec, B. K.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Navarro, J. F.
    Alexeyenko, A.
    Reimegård, J.
    McKee, Lauren S.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Mannapperuma, C.
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience. University of Adelaide, Australia.
    Ståhl, P. L.
    Sundström, J. F.
    Street, N. R.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatially resolved transcriptome profiling in model plant species2017In: Nature Plants, ISSN 2055-0278, Vol. 3, article id 17061Article in journal (Refereed)
    Abstract [en]

    Understanding complex biological systems requires functional characterization of specialized tissue domains. However, existing strategies for generating and analysing high-throughput spatial expression profiles were developed for a limited range of organisms, primarily mammals. Here we present the first available approach to generate and study high-resolution, spatially resolved functional profiles in a broad range of model plant systems. Our process includes high-throughput spatial transcriptome profiling followed by spatial gene and pathway analyses. We first demonstrate the feasibility of the technique by generating spatial transcriptome profiles from model angiosperms and gymnosperms microsections. In Arabidopsis thaliana we use the spatial data to identify differences in expression levels of 141 genes and 189 pathways in eight inflorescence tissue domains. Our combined approach of spatial transcriptomics and functional profiling offers a powerful new strategy that can be applied to a broad range of plant species, and is an approach that will be pivotal to answering fundamental questions in developmental and evolutionary biology.

  • 115. Gioti, Anastasia
    et al.
    Nystedt, Björn
    Li, Wenjun
    Xu, Jun
    Andersson, Anna
    Averette, Anna F.
    Muench, Karin
    Wang, Xuying
    Kappauf, Catharine
    Kingsbury, Joanne M.
    Kraak, Bart
    Walker, Louise A.
    Johansson, Henrik J.
    Holm, Tina
    Lehtiö, Janne
    Stajich, Jason E.
    Mieczkowski, Piotr
    Kahmann, Regine
    Kennell, John C.
    Cardenas, Maria E.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Saunders, Charles W.
    Boekhout, Teun
    Dawson, Thomas L.
    Munro, Carol A.
    de Groot, Piet W. J.
    Butler, Geraldine
    Heitman, Joseph
    Scheynius, Annika
    Genomic Insights into the Atopic Eczema-Associated Skin Commensal Yeast Malassezia sympodialis2013In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 4, no 1, p. e00572-12-Article in journal (Refereed)
    Abstract [en]

    Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e. g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci. IMPORTANCE Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

  • 116. Granholm, V.
    et al.
    Kim, S.
    Fernandez Navarro, José
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sjölund, E.
    Smith, R. D.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, Centres, SeRC - Swedish e-Science Research Centre.
    Fast and accurate database searches with MS-GF+percolator2014In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 13, no 2, p. 890-897Article in journal (Refereed)
    Abstract [en]

    One can interpret fragmentation spectra stemming from peptides in mass-spectrometry-based proteomics experiments using so-called database search engines. Frequently, one also runs post-processors such as Percolator to assess the confidence, infer unique peptides, and increase the number of identifications. A recent search engine, MS-GF+, has shown promising results, due to a new and efficient scoring algorithm. However, MS-GF+ provides few statistical estimates about the peptide-spectrum matches, hence limiting the biological interpretation. Here, we enabled Percolator processing for MS-GF+ output and observed an increased number of identified peptides for a wide variety of data sets. In addition, Percolator directly reports p values and false discovery rate estimates, such as q values and posterior error probabilities, for peptide-spectrum matches, peptides, and proteins, functions that are useful for the whole proteomics community.

  • 117. Granholm, Viktor
    et al.
    Fernandez Navarro, José
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Noble, William Stafford
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Determining the calibration of confidence estimation procedures for unique peptides in shotgun proteomics2013In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 80, p. 123-131Article in journal (Refereed)
    Abstract [en]

    The analysis of a shotgun proteomics experiment results in a list of peptide-spectrum matches (PSMs) in which each fragmentation spectrum has been matched to a peptide in a database. Subsequently, most protein inference algorithms rank peptides according to the best-scoring PSM for each peptide. However, there is disagreement in the scientific literature on the best method to assess the statistical significance of the resulting peptide identifications. Here, we use a previously described calibration protocol to evaluate the accuracy of three different peptide-level statistical confidence estimation procedures: the classical Fisher's method, and two complementary procedures that estimate significance, respectively, before and after selecting the top-scoring PSM for each spectrum. Our experiments show that the latter method, which is employed by MaxQuant and Percolator, produces the most accurate, well-calibrated results.

  • 118. Granholm, Viktor
    et al.
    Noble, William Stafford
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    A cross-validation scheme for machine learning algorithms in shotgun proteomics2012In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 13, p. S3-Article in journal (Refereed)
    Abstract [en]

    Peptides are routinely identified from mass spectrometry-based proteomics experiments by matching observed spectra to peptides derived from protein databases. The error rates of these identifications can be estimated by target-decoy analysis, which involves matching spectra to shuffled or reversed peptides. Besides estimating error rates, decoy searches can be used by semi-supervised machine learning algorithms to increase the number of confidently identified peptides. As for all machine learning algorithms, however, the results must be validated to avoid issues such as overfitting or biased learning, which would produce unreliable peptide identifications. Here, we discuss how the target-decoy method is employed in machine learning for shotgun proteomics, focusing on how the results can be validated by cross-validation, a frequently used validation scheme in machine learning. We also use simulated data to demonstrate the proposed cross-validation scheme's ability to detect overfitting.

  • 119. Green, Anna
    et al.
    Green, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Linkoping University, Sweden.
    Rehnberg, Malin
    Svensson, Anneli
    Gunnarsson, Cecilia
    Jonasson, Jon
    Assessment of HaloPlex Amplification for Sequence Capture and Massively Parallel Sequencing of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Genes2015In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 17, no 1, p. 31-42Article in journal (Refereed)
    Abstract [en]

    The genetic basis of arrhythmogenic right ventricular cardiomyopathy (ARVC) is complex. Mutations in genes encoding components of the cardiac desmosomes have been implicated as being causally related to ARVC. Next-generation sequencing allows parallel sequencing and duplication/deletion analysis of many genes simultaneously, which is appropriate for screening of mutations in disorders with heterogeneous genetic backgrounds. We designed and validated a next-generation sequencing test panel for ARVC using HaloPlex. We used SureDesign to prepare a HaloPlex enrichment system for sequencing of DES, DSC2, DSG2, DSP, JUP, PKP2, RYR2, TGFB3, TMEM43, and TIN from patients with ARVC using a MiSeq instrument. Performance characteristics were determined by comparison with Sanger, as the gold standard, and TruSeq Custom Amplicon sequencing of DSC2, DSG2, DSP, JUP, and PKP2. All the samples were successfully sequenced after HaloPlex capture, with >99% of targeted nucleotides covered by >20x. The sequences were of high quality, although one problematic area due to a presumptive context-specific sequencing error causing motif Located in exon 1 of the DSP gene was detected. The mutations found by Sanger sequencing were also found using the HaloPlex technique. Depending on the bioinformatics pipeline, sensitivity varied from 99.3% to 100%, and specificity varied from 99.90/0 to 100%. Three variant positions found by Sanger and HaloPlex sequencing were missed by TruSeq Custom Amplicon owing to Loss of coverage.

  • 120.
    Green, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Linköping University, Sweden; National Board of Forensic Medicine, Sweden.
    Hasmats, Johanna
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kupershmidt, Ilya
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. NextBio, USA.
    Edsgärd, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    de Petris, Luigi
    Lewensohn, Rolf
    Blackhall, Fiona
    Vikingsson, Svante
    Besse, Benjamin
    Lindgren, Andrea
    Branden, Eva
    Koyi, Hirsh
    Peterson, Curt
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Using Whole-Exome Sequencing to Identify Genetic Markers for Carboplatin and Gemcitabine-Induced Toxicities2016In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 22, no 2, p. 366-373Article in journal (Refereed)
    Abstract [en]

    Purpose: Chemotherapies are associated with significant interindividual variability in therapeutic effect and adverse drug reactions. In lung cancer, the use of gemcitabine and carboplatin induces grade 3 or 4 myelosuppression in about a quarter of the patients, while an equal fraction of patients is basically unaffected in terms of myelosuppressive side effects. We therefore set out to identify genetic markers for gemcitabine/carboplatin-induced myelosuppression. Experimental Design: We exome sequenced 32 patients that suffered extremely high neutropenia and thrombocytopenia (grade 3 or 4 after first chemotherapy cycle) or were virtually unaffected (grade 0 or 1). The genetic differences/polymorphism between the groups were compared using six different bioinformatics strategies: (i) whole-exome nonsynonymous single-nucleotide variants association analysis, (ii) deviation from Hardy-Weinberg equilibrium, (iii) analysis of genes selected by a priori biologic knowledge, (iv) analysis of genes selected from gene expression meta-analysis of toxicity datasets, (v) Ingenuity Pathway Analysis, and (vi) FunCoup network enrichment analysis. Results: A total of 53 genetic variants that differed among these groups were validated in an additional 291 patients and were correlated to the patients' myelosuppression. In the validation, we identified rs1453542 in OR4D6 (P = 0.0008; OR, 5.2; 95% CI, 1.8-18) as a marker for gemcitabine/carboplatin-induced neutropenia and rs5925720 in DDX53 (P = 0.0015; OR, 0.36; 95% CI, 0.17-0.71) as a marker for thrombocytopenia. Patients homozygous for the minor allele of rs1453542 had a higher risk of neutropenia, and for rs5925720 the minor allele was associated with a lower risk for thrombocytopenia. Conclusions: We have identified two new genetic markers with the potential to predict myelosuppression induced by gemcitabine/ carboplatin chemotherapy.

  • 121.
    Green, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Khan, Muhammad Suleman
    Jakobsen-Falk, Ingrid
    Avall-Lundqvist, Elisabeth
    Peterson, Curt
    Impact of CYP3A5(*)3 and CYP2C8-HapC on Paclitaxel/Carboplatin-Induced Myelosuppression in Patients with Ovarian Cancer2011In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 100, no 10, p. 4205-4209Article in journal (Refereed)
    Abstract [en]

    The influence of genetic variants on paclitaxel-induced toxicity is of considerable interest for reducing adverse drug reactions. Recently, the genetic variants CYP2C8(*)3, CYP2C8-HapC, and CYP3A5(*)3 were associated with paclitaxel-induced neurotoxicity. We, therefore, investigated the impact of CYP2C8-HapC and CYP3A5(*)3 on paclitaxel/carboplatin-induced myelosuppression and neurotoxicity. Thirty-three patients from a prospective pharmacokinetics study were genotyped using pyrosequencing. Patients with variant alleles of CYP2C8-HapC were found to have significantly lower nadir values of both leukocytes and neutrophils (p < 0.05) than patients with the wild-type genotype. CYP3A5(*)3/(*)1 patients were shown to have borderline, significantly lower nadir values of leukocytes (p = 0.07) than (*)3/(*)3 patients. Combining the two genotypes resulted in a significant correlation with both leukopenia and neutropenia (p = 0.01). No effect of these genetic variants on neurotoxicity could be shown in this rather small study, but their importance for paclitaxel-induced toxicity could be confirmed.

  • 122.
    Green, Henrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Stål, O.
    Bachmeier, K.
    Bäcklund, L. M.
    Carlsson, L.
    Hansen, J.
    Lagerlund, M.
    Norberg, B.
    Franzén, Å.
    Åleskog, A.
    Malmström, A.
    Pegylated liposomal doxorubicin as first-line monotherapy in elderly women with locally advanced or metastatic breast cancer: Novel treatment predictive factors identified2011In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 313, no 2, p. 145-153Article in journal (Refereed)
    Abstract [en]

    We investigated the efficacy and safety of single-agent pegylated liposomal doxorubicin (PLD) as first-line treatment for elderly women with advanced breast cancer and evaluated predictive markers for response and toxicity. Twenty-five women >= 65 years received 40 mg/m(2) PLD every 28 days. Time to treatment failure (TTF), response rate, time to progression (TTP) and overall survival (OS) was calculated. The ABCB1 single nucleotide polymorphisms (SNP), tumor MRN complex, and TOPOII alpha were analyzed. A mean of 7.4 cycles PLD were administered and TIT was 5.5 months and OS 20.6 months. ABCB1 SNPs were found to correlate to both efficacy and toxicity, while tumor expression of the MRN complex and TOPOII alpha correlated to TTP. PLD is a safe and effective treatment for elderly breast cancer patients. Also potential predictive markers were identified.

  • 123. Gregers, J.
    et al.
    Green, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Division of Drug Research, Department of Medical and Health Sciences, Linköpings Universitet, Linköping, Sweden.
    Christensen, I. J.
    Dalhoff, K.
    Schroeder, H.
    Carlsen, N.
    Rosthoej, S.
    Lausen, B.
    Schmiegelow, K.
    Peterson, C.
    Polymorphisms in the ABCB1 gene and effect on outcome and toxicity in childhood acute lymphoblastic leukemia2015In: The Pharmacogenomics Journal, ISSN 1470-269X, E-ISSN 1473-1150, Vol. 15, no 4, p. 372-379Article in journal (Refereed)
    Abstract [en]

    The membrane transporter P-glycoprotein, encoded by the ABCB1 gene, influences the pharmacokinetics of anti-cancer drugs. We hypothesized that variants of ABCB1 affect outcome and toxicity in childhood acute lymphoblastic leukemia (ALL). We studied 522 Danish children with ALL, 93% of all those eligible. Risk of relapse was increased 2.9-fold for patients with the 1199GA variant versus 1199GG (P = 0.001), and reduced 61% and 40%, respectively, for patients with the 3435CT or 3435TT variants versus 3435CC (overall P = 0.02). The degree of bone marrow toxicity during doxorubicin, vincristine and prednisolone induction therapy was more prominent in patients with 3435TT variant versus 3435CT/3435CC (P = 0.01/P < 0.0001). We observed more liver toxicity after high-dose methotrexate in patients with 3435CC variant versus 3435CT/TT ( P = 0.03). In conclusion, there is a statistically significant association between ABCB1 polymorphisms, efficacy and toxicity in the treatment of ALL, and ABCB1 1199G > A may be a new possible predictive marker for outcome in childhood ALL.

  • 124. Grskovic, Branka
    et al.
    Zrnec, Dario
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Popovic, Maja
    Mrsic, Gordan
    DNA methylation: the future of crime scene investigation?2013In: Molecular Biology Reports, ISSN 0301-4851, E-ISSN 1573-4978, Vol. 40, no 7, p. 4349-4360Article in journal (Refereed)
    Abstract [en]

    Proper detection and subsequent analysis of biological evidence is crucial for crime scene reconstruction. The number of different criminal acts is increasing rapidly. Therefore, forensic geneticists are constantly on the battlefield, trying hard to find solutions how to solve them. One of the essential defensive lines in the fight against the invasion of crime is relying on DNA methylation. In this review, the role of DNA methylation in body fluid identification and other DNA methylation applications are discussed. Among other applications of DNA methylation, age determination of the donor of biological evidence, analysis of the parent-of-origin specific DNA methylation markers at imprinted loci for parentage testing and personal identification, differentiation between monozygotic twins due to their different DNA methylation patterns, artificial DNA detection and analyses of DNA methylation patterns in the promoter regions of circadian clock genes are the most important ones. Nevertheless, there are still a lot of open chapters in DNA methylation research that need to be closed before its final implementation in routine forensic casework.

  • 125. Grubisic, Lorena M.
    et al.
    Bertilsson, Stefan
    Eiler, Alexander
    Heinrich, Friederike
    Brutemark, Andreas
    Alonso-Saez, Laura
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Gantner, Stephan
    Riemann, Lasse
    Beier, Sara
    Lake bacterioplankton dynamics over diurnal timescales2017In: Freshwater Biology, ISSN 0046-5070, E-ISSN 1365-2427, Vol. 62, no 1, p. 191-204Article in journal (Refereed)
    Abstract [en]

    1. Planktonic bacterial community dynamics over short timescales can be of great importance for food webs and ecosystem functioning but are rarely described when microbial community and composition are assessed. To study the significance of such dynamics we sampled the surface water at the deepest point of a mesotrophic lake (Lake Erken, Sweden) every third hour over two days. 2. By combining 454 pyrosequencing of 16S rRNA genes with bromodeoxyuridine immunocapturing of DNA, replicating populations were identified and compared to the community retrieved from total DNA samples. This comparison revealed a significant difference between the actively replicating and total community. 3. The high-frequency diurnal sampling was compared to a year-long survey conducted in the same lake in order to compare the diurnal and seasonal variation in bacterioplankton community composition. At the diurnal-scale, the variation was significantly higher in the replicating than in the total community. However, variation in both active and total diurnal community was significantly lower than the variation in the seasonal total community. 4. Our analysis revealed pronounced short-term dynamics of individual bacterial populations uncoupled from the diurnal light cycle. For example, the proliferating fraction of the most abundant bacterial tribe (LD12) followed a cyclic pattern that covaried with viral abundance. This implies that environmental factors other than light may act as important drivers of microbial community composition, at least in mesotrophic Lake Erken.

  • 126. Grunewald, J.
    et al.
    Kaiser, Y.
    Ostadkarampour, M.
    Rivera, N. V.
    Vezzi, F.
    Lötstedt, B.
    Olsen, R. -A
    Sylwan, L.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sandalova, T.
    Ahlgren, K. M.
    Wahlström, J.
    Achour, A.
    Ronninger, M.
    Eklund, A.
    T-cell receptor-HLA-DRB1 associations suggest specific antigens in pulmonary sarcoidosis2016In: European Respiratory Journal, ISSN 0903-1936, E-ISSN 1399-3003, Vol. 47, no 3, p. 898-909Article in journal (Refereed)
    Abstract [en]

    In pulmonary sarcoidosis, CD4+; T-cells expressing T-cell receptor Vα2.3 accumulate in the lungs of HLA-DRB1∗03+; patients. To investigate T-cell receptor-HLA-DRB1∗03 interactions underlying recognition of hitherto unknown antigens, we performed detailed analyses of T-cell receptor expression on bronchoalveolar lavage fluid CD4+; T-cells from sarcoidosis patients. Pulmonary sarcoidosis patients (n=43) underwent bronchoscopy with bronchoalveolar lavage. T-cell receptor α and β chains of CD4+; T-cells were analysed by flow cytometry, DNA-sequenced, and threedimensional molecular models of T-cell receptor-HLA-DRB1∗03 complexes generated. Simultaneous expression of Vα2.3 with the Vβ22 chain was identified in the lungs of all HLADRB1∗ 03+; patients. Accumulated Vα2.3/Vβ22-expressing T-cells were highly clonal, with identical or near-identical Vα2.3 chain sequences and inter-patient similarities in Vβ22 chain amino acid distribution. Molecular modelling revealed specific T-cell receptor-HLA-DRB1∗03-peptide interactions, with a previously identified, sarcoidosis-associated vimentin peptide, (Vim)429-443 DSLPLVDTHSKRTLL, matching both the HLA peptide-binding cleft and distinct T-cell receptor features perfectly. We demonstrate, for the first time, the accumulation of large clonal populations of specific Vα2.3/Vβ22 T-cell receptor-expressing CD4+; T-cells in the lungs of HLA-DRB1∗03+; sarcoidosis patients. Several distinct contact points between Vα2.3/Vβ22 receptors and HLA-DRB1∗03 molecules suggest presentation of prototypic vimentin-derived peptides.

  • 127. Guo, Songchang
    et al.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Su, Jianping
    Zhang, Qian
    Qi, Delin
    Zhou, Jie
    Zhong, Yang
    Zhao, Xinquan
    Liu, Jianquan
    Origin of mitochondrial DNA diversity of domestic yaks2006In: BMC Evolutionary Biology, ISSN 1471-2148, E-ISSN 1471-2148, Vol. 6Article in journal (Refereed)
    Abstract [en]

    Background: The domestication of plants and animals was extremely important anthropologically. Previous studies have revealed a general tendency for populations of livestock species to include deeply divergent maternal lineages, indicating that they were domesticated in multiple, independent events from genetically discrete wild populations. However, in water buffalo, there are suggestions that a similar deep maternal bifurcation may have originated from a single population. These hypotheses have rarely been rigorously tested because of a lack of sufficient wild samples. To investigate the origin of the domestic yak (Poephagus grunnies), we analyzed 637 bp of maternal inherited mtDNA from 13 wild yaks (including eight wild yaks from a small population in west Qinghai) and 250 domesticated yaks from major herding regions. Results: The domestic yak populations had two deeply divergent phylogenetic groups with a divergence time of > 100,000 yrs BP. We here show that haplotypes clustering with two deeply divergent maternal lineages in domesticated yaks occur in a single, small, wild population. This finding suggests that all domestic yaks are derived from a single wild gene pool. However, there is no clear correlation of the mtDNA phylogenetic clades and the 10 morphological types of sampled yaks indicating that the latter diversified recently. Relatively high diversity was found in Qinghai and Tibet around the current wild distribution, in accordance with previous suggestions that the earliest domestications occurred in this region. Conventional molecular clock estimation led to an unrealistic early dating of the start of the domestication. However, Bayesian estimation of the coalescence time allowing a relaxation of the mutation rate Conclusion: The information gathered here and the previous studies of other animals show that the demographic histories of domestication of livestock species were highly diverse despite the common general feature of deeply divergent maternal lineages. The results further suggest that domestication of local wild prey ungulate animals was a common occurrence during the development of human civilization following the postglacial colonization in different locations of the world, including the high, arid Qinghai-Tibetan Plateau.

  • 128. Gvozdik, Vaclav
    et al.
    Moravec, Jiri
    Klütsch, Cornelya
    KTH, School of Biotechnology (BIO), Gene Technology.
    Kotlik, Petr
    Phylogeography of the Middle Eastern tree frogs (Hyla, Hylidae, Amphibia) as inferred from nuclear and mitochondrial DNA variation, with a description of a new species2010In: Molecular Phylogenetics and Evolution, ISSN 1055-7903, E-ISSN 1095-9513, Vol. 55, no 3, p. 1146-1166Article in journal (Refereed)
    Abstract [en]

    Evolutionary relationships of the tree frogs from the Middle East and the demographic histories of their populations were studied using a combination of mitochondrial and nuclear genes. Hyla savignyi and neighboring populations of H. orientalis (former eastern populations of H. arborea) were the main focus taxa. Within H. savignyi, a deep phylogenetic divergence dated about 8.4 Ma was discovered. Southern populations from Yemen, Jordan, southern Syria and extreme north-eastern Israel are hereby described as a new species, H. felixarabica sp. nov. Our study points to a biogeographic connection of the south-western Arabian Peninsula and southern Levant and to the importance of the Dead Sea Rift as a historical barrier geographically separating the new species from H. savignyi. Major genetic breaks revealed within species (H. felixarabica: Yemen vs. Jordan-Syria; H. savignyi sensu stricto: Levant vs. Turkey-Iran) are probably connected to climate changes during the Plio-Pleistocene boundary, while the finer phylogeographic structuring probably resulted from the Quaternary climate oscillations. The Cypriote population of H. savignyi originated from southern Anatolia relatively recently. Hyla orientalis from the southern Black Sea region seems to be genetically quite uniform, although two phylogeographic units with western Turkish and Caucasus-Caspian affinities might be detected. Hyla savignyi and H. orientalis carry signals of population expansions dated to the middle to late Pleistocene, while populations of H. felixarabica seem to have rather been constant in size, which might indicate more stable climatic conditions in the southern regions during the Quaternary.

  • 129.
    Gyarmati, Peter
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Song, Yajing
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hällman, Jimmie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Chemical fragmentation for massively parallel sequencing library preparation2013In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 168, no 1, p. 95-100Article in journal (Refereed)
    Abstract [en]

    Fragmentation is essential in most library preparation protocols for use with massively parallel sequencing systems. Complexes that generate hydroxyl radicals, such as iron-EDTA, can be used to introduce random DNA cleavage. Here we describe a chemical fragmentation method that can be incorporated into library preparation protocols for next-generation sequencing workflows. This protocol has been validated by whole genome, amplicon and exome sequencing. Chemical fragmentation is a cost-effective alternative to current fragmentation methods that has no observable sequence bias and requires no instrumentation.

  • 130.
    Hasmats, Johanna
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Analysis of genetic variations in cancer2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The aim of this thesis is to apply recently developed technologies for genomic variation analyses, and to ensure quality of the generated information for use in preclinical cancer research.

    Faster access to a patients’ full genomic sequence for a lower cost makes it possible for end users such as clinicians and physicians to gain a more complete understanding of the disease status of a patient and adjust treatment accordingly. Correct biological interpretation is important in this context, and can only be provided through fast and simple access to relevant high quality data.

    Therefore, we here propose and validate new bioinformatic strategies for biomarker selection for prediction of response to cancer therapy. We initially explored the use of bioinformatic tools to select interesting targets for toxicity in carboplatin and paclitaxel on a smaller scale. From our findings we then further extended the analysis to the entire exome to look for biomarkers as targets for adverse effects from carboplatin and gemcitabine. To investigate any bias introduced by the methods used for targeting the exome, we analyzed the mutation profiles in cancer patients by comparing whole genome amplified DNA to unamplified DNA. In addition, we applied RNA-seq to the same patients to further validate the variations obtained by sequencing of DNA. The understanding of the human cancer genome is growing rapidly, thanks to methodological development of analysis tools. The next step is to implement these tools as a part of a chain from diagnosis of patients to genomic research to personalized treatment.

  • 131.
    Hasmats, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Orear, Cedric
    Validire, Pierre
    Huss, Mikael
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Assessment of Whole Genome Amplification for Sequence Capture and Massively Parallel Sequencing2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, p. e84785-Article in journal (Refereed)
    Abstract [en]

    Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.

  • 132.
    Hasmats, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Solnestam, Beata Werne
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zajac, Pawel
    Huss, Mikael
    Orear, Cedric
    Validire, Pierre
    Bjursell, Magnus
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 425, no 2, p. 379-383Article in journal (Refereed)
    Abstract [en]

    Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a 929 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.

  • 133.
    Hasmats, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kupershmidt, Ilya
    Edsgärd, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    de Petris, Luigi
    Lewensohn, Rolf
    Alexeyenko, Andrey
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Blackhall, Fiona
    Bess, Benjamin
    Lindgren, Andrea
    Sörenson, Sverre
    Brandén, Eva
    Koyi, Hirsh
    Peterson, Curt
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Using whole exome sequencing to identify genetic candidates for carboplatin and gemcitabine induced toxicitiesArticle in journal (Other academic)
    Abstract [en]

    Chemotherapies are associated with significant inter-individual variability in therapeutic effect and adverse drug reactions. In lung cancer the use of gemcitabine and carboplatin induces grade 3-4 myelosuppression in about ¼ of the patients while an equal fraction of patients are basically unaffected in terms of myelosuppressive side effects. We therefore set out to try to identify genetic markers for gemcitabine / carboplatin induced myelosuppression. We selected 32 patients that suffered extremely high neutropenia and thrombocytopenia (grade 3 or 4 after first chemotherapy cycle) or were virtually unaffected (grade 0-1 after the first chemotherapy cycle) by the chemotherapy out of 243 lung cancer patients treated with gemcitabine / carboplatin. These patients were exome sequenced and their genetic differences compared using six different bioinformatic strategies; whole exome non-synonymous SNV association analysis, deviation from Hardy-Weinberg equilibrium, analysis of genes selected by a priori biological knowledge, analysis of genes selected from gene expression meta-analysis of toxicity data sets, Ingenuity pathway analysis and FunCoup network enrichment analysis. All patients were successfully sequenced and 5000-7000 non-synonymous single nucleotide variants were identified in each patient. PI3 (elastase specific inhibitor in neutrophils) showed the strongest association in the single SNV analysis (nominal p=0.0005). Further, variants within IL37, an inhibitor of the innate immune system, and CSAG1, a tumor antigen, differed among the two patient groups and appeared among the top hits in several of the performed analysis, indicating that the approach identifies genetic variants associated with the immune system and tumor differentiation, which might be important for the sensitivity to chemotherapeutic agents. However, the associations reported here are in a need of replication before clinical interpretations can be made.

  • 134.
    Hasmats, Johanna
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kupershmidt, Ilya
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rodriguez-Antona, Cristina
    Su, Qiaojuan Jane
    Khan, Muhammad Suleman
    Jara, Carlos
    Mielgo, Xabier
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gréen, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues2012In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 506, no 1, p. 62-68Article in journal (Refereed)
    Abstract [en]

    The growing collection of publicly available high-throughput data provides an invaluable resource for generating preliminary in silico data in support of novel hypotheses. In this study we used a cross-dataset meta-analysis strategy to identify novel candidate genes and genetic variations relevant to paclitaxel/carboplatin-induced myelosuppression and neuropathy. We identified genes affected by drug exposure and present in tissues associated with toxicity. From ten top-ranked genes 42 non-synonymous single nucleotide polymorphisms (SNPs) were identified in silico and genotyped in 94 cancer patients treated with carboplatin/paclitaxel. We observed variations in 11 SNPs, of which seven were present in a sufficient frequency for statistical evaluation. Of these seven SNPs. three were present in ABCA1 and ATM, and showed significant or borderline significant association with either myelosuppression or neuropathy. The strikingly high number of associations between genotype and clinically observed toxicity provides support for our data-driven computations strategy to identify biomarkers for drug toxicity.

  • 135. Hedskog, Charlotte
    et al.
    Mild, Mattias
    Jernberg, Johanna
    Sherwood, Ellen
    Bratt, Göran
    Leitner, Thomas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson, Björn
    Albert, Jan
    Dynamics of HIV-1 Quasispecies during Antiviral Treatment Dissected Using Ultra-Deep Pyrosequencing2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 7, p. e11345-Article in journal (Refereed)
    Abstract [en]

    Background: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. Principal Findings: Here, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180-220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406-35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07-0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. Conclusions: With this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.

  • 136. Herlemann, Daniel P. R.
    et al.
    Labrenz, Matthias
    Juergens, Klaus
    Bertilsson, Stefan
    Waniek, Joanna J.
    Andersson, Anders F.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Transitions in bacterial communities along the 2000 km salinity gradient of the Baltic Sea2011In: ISME JOURNAL, ISSN 1751-7362, Vol. 5, no 10, p. 1571-1579Article in journal (Refereed)
    Abstract [en]

    Salinity is a major factor controlling the distribution of biota in aquatic systems, and most aquatic multicellular organisms are either adapted to life in saltwater or freshwater conditions. Consequently, the saltwater-freshwater mixing zones in coastal or estuarine areas are characterized by limited faunal and floral diversity. Although changes in diversity and decline in species richness in brackish waters is well documented in aquatic ecology, it is unknown to what extent this applies to bacterial communities. Here, we report a first detailed bacterial inventory from vertical profiles of 60 sampling stations distributed along the salinity gradient of the Baltic Sea, one of world's largest brackish water environments, generated using 454 pyrosequencing of partial (400 bp) 16S rRNA genes. Within the salinity gradient, bacterial community composition altered at broad and finer-scale phylogenetic levels. Analogous to faunal communities within brackish conditions, we identified a bacterial brackish water community comprising a diverse combination of freshwater and marine groups, along with populations unique to this environment. As water residence times in the Baltic Sea exceed 3 years, the observed bacterial community cannot be the result of mixing of fresh water and saltwater, but our study represents the first detailed description of an autochthonous brackish microbiome. In contrast to the decline in the diversity of multicellular organisms, reduced bacterial diversity at brackish conditions could not be established. It is possible that the rapid adaptation rate of bacteria has enabled a variety of lineages to fill what for higher organisms remains a challenging and relatively unoccupied ecological niche.

  • 137. Herlemann, Daniel P. R.
    et al.
    Lundin, Daniel
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Labrenz, Matthias
    Juergens, Klaus
    Phylogenetic Signals of Salinity and Season in Bacterial Community Composition Across the Salinity Gradient of the Baltic Sea2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 1883Article in journal (Refereed)
    Abstract [en]

    Understanding the key processes that control bacterial community composition has enabled predictions of bacterial distribution and function within ecosystems. In this study, we used the Baltic Sea as a model system to quantify the phylogenetic signal of salinity and season with respect to bacterioplankton community composition. The abundances of 16S rRNA gene amplicon sequencing reads were analyzed from samples obtained from similar geographic locations in July and February along a brackish to marine salinity gradient in the Baltic Sea. While there was no distinct pattern of bacterial richness at different salinities, the number of bacterial phylotypes in winter was significantly higher than in summer. Bacterial community composition in brackish vs. marine conditions, and in July vs. February was significantly different. Non-metric multidimensional scaling showed that bacterial community composition was primarily separated according to salinity and secondly according to seasonal differences at all taxonomic ranks tested. Similarly, quantitative phylogenetic clustering implicated a phylogenetic signal for both salinity and seasonality. Our results suggest that global patterns of bacterial community composition with respect to salinity and season are the result of phylogenetically clustered ecological preferences with stronger imprints from salinity.

  • 138. Herlemann, Daniel P. R.
    et al.
    Lundin, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Labrenz, Matthias
    Jürgens, Klaus
    Zheng, Zongli
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria2013In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 4, no 3, p. e00569-12-Article in journal (Refereed)
    Abstract [en]

    The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype's distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates. IMPORTANCE The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem.

  • 139. Hertzberg, M
    et al.
    Aspeborg, H
    Schrader, J
    Andersson, Anders
    KTH, School of Biotechnology (BIO), Gene Technology.
    Erlandsson, R
    Blomqvist, K
    Bhalerao, R
    Uhlen, M
    Teeri, T T
    Lundeberg, J
    Sundberg, B
    Nilsson, P
    Sandberg, G
    A transcriptional roadmap to wood formation2001In: Proc Natl Acad Sci U S A, Vol. 98, no 25, p. 14732-14737Article in journal (Refereed)
    Abstract [en]

    The large vascular meristem of poplar trees with its highly organized secondary xylem enables the boundaries between different developmental zones to be easily distinguished. This property of wood-forming tissues allowed us to determine a unique tissue-specific transcript profile for a well defined developmental gradient. RNA was prepared from different developmental stages of xylogenesis for DNA microarray analysis by using a hybrid aspen unigene set consisting of 2,995 expressed sequence tags. The analysis revealed that the genes encoding lignin and cellulose biosynthetic enzymes, as well as a number of transcription factors and other potential regulators of xylogenesis, are under strict developmental stage-specific transcriptional regulation.

  • 140. Hillbertz, Nicolette H. C. Salmon
    et al.
    Isaksson, Magnus
    Karlsson, Elinor K.
    Hellmen, Eva
    Pielberg, Gerli Rosengren
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Wade, Claire M.
    Von Euler, Henrik
    Gustafson, Ulla
    Hedhammar, Ake
    Nilsson, Mats
    Lindblad-Toh, Kerstin
    Andersson, Leif
    Andersson, Goran
    Duplication of FGF3, FGF4, FGF19 and ORAOV1 causes hair ridge and predisposition to dermoid sinus in Ridgeback dogs2007In: Nature Genetics, ISSN 1061-4036, E-ISSN 1546-1718, Vol. 39, no 11, p. 1318-1320Article in journal (Refereed)
    Abstract [en]

    The dorsal hair ridge in Rhodesian and Thai Ridgeback dogs is caused by a dominant mutation that also predisposes to the congenital developmental disorder dermoid sinus. Here we show that the causative mutation is a 133-kb duplication involving three fibroblast growth factor (FGF) genes. FGFs play a crucial role in development, suggesting that the ridge and dermoid sinus are caused by dysregulation of one or more of the three FGF genes during development.

  • 141. Hofmeister, Wolfgang
    et al.
    Nilsson, Daniel
    Topa, Alexandra
    Anderlid, Britt-Marie
    Darki, Fahimeh
    Matsson, Hans
    Paez, Isabel Tapia
    Klingberg, Torkel
    Samuelsson, Lena
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO), Gene Technology.
    Vezzi, Francesco
    Kere, Juha
    Nordenskjold, Magnus
    Lundberg, Elisabeth Syk
    Lindstrand, Anna
    CTNND2: a candidate gene for reading problems and mild intellectual disability2015In: J MED GENET, ISSN 0022-2593, Vol. 52, no 2, p. 111-122Article in journal (Refereed)
    Abstract [en]

    Background Cytogenetically visible chromosomal translocations are highly informative as they can pinpoint strong effect genes even in complex genetic disorders. Methods and results Here, we report a mother and daughter, both with borderline intelligence and learning problems within the dyslexia spectrum, and two apparently balanced reciprocal translocations: t(1;8)(p22; q24) and t(5; 18)(p15; q11). By low coverage mate-pair whole-genome sequencing, we were able to pinpoint the genomic breakpoints to 2 kb intervals. By direct sequencing, we then located the chromosome 5p breakpoint to intron 9 of CTNND2. An additional case with a 163 kb microdeletion exclusively involving CTNND2 was identified with genome-wide array comparative genomic hybridisation. This microdeletion at 5p15.2 is also present in mosaic state in the patient's mother but absent from the healthy siblings. We then investigated the effect of CTNND2 polymorphisms on normal variability and identified a polymorphism (rs2561622) with significant effect on phonological ability and white matter volume in the left frontal lobe, close to cortical regions previously associated with phonological processing. Finally, given the potential role of CTNND2 in neuron motility, we used morpholino knockdown in zebrafish embryos to assess its effects on neuronal migration in vivo. Analysis of the zebrafish forebrain revealed a subpopulation of neurons misplaced between the diencephalon and telencephalon. Conclusions Taken together, our human genetic and in vivo data suggest that defective migration of subpopulations of neuronal cells due to haploinsufficiency of CTNND2 contribute to the cognitive dysfunction in our patients.

  • 142. Hoiom, Veronica
    et al.
    Tuominen, Rainer
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology.
    Linden, Diana
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mansson-Brahme, Eva
    Egyhazi, Suzanne
    Sjöberg, Klas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Hansson, Johan
    MC1R variation and melanoma risk in the Swedish population in relation to clinical and pathological parameters2009Article in journal (Refereed)
    Abstract [en]

    The genetic background of cutaneous malignant melanoma (CMM) includes both germ line aberrations in high-penetrance genes, like CDKN2A, and allelic variation in low-penetrance genes like the melanocortin-1 receptor gene, MC1R. Red-hair colour associated MC1R alleles (RHC) have been associated with red hair, fair skin and risk of CMM. We investigated MC1R and CDKN2A variation in relation to phenotype, clinical factors and CMM risk in the Swedish population. The study cohort consisted of sporadic primary melanoma patients, familial melanoma patients and a control group. An allele-dose dependent increase in melanoma risk for carriers of variant MC1R alleles (after adjusting for phenotype), with an elevated risk among familial CMM patients, was observed. This elevated risk was found to be significantly associated with an increased frequency of dysplastic nevi (DN) among familial patients compared to sporadic patients. MC1R variation was found to be less frequent among acral lentiginous melanomas (ALM) and dependent on tumour localisation. No association was found between CDKN2A gene variants and general melanoma risk. Two new variants in the POMC gene were identified in red haired individuals without RHC alleles.

  • 143. Holmberg, A.
    et al.
    Blomstergren, A.
    Nord, O.
    Lukacs, M.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures2005In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 26, no 3, p. 501-510Article in journal (Refereed)
    Abstract [en]

    The biotin-streptavidin system is the strongest noncovalent biological interaction known, having a dissociation constant, K-d, in the order of 4 x 10(-14) m. The strength and specificity of the interaction has led it to be one of the most widely used affinity pairs in molecular, immunological, and cellular assays. However, it has previously been impossible to re-use any streptavidin solid support, since the conditions needed to break the interaction with biotin has led to the denaturation of the streptavidin. Here, we show that a short incubation in nonionic aqueous solutions at temperatures above 70degreesC can efficiently break the interaction without denaturing the streptavidin tetramer. Both biotin and the streptavidin remain active after dissociation and both molecules can therefore be re-used. The efficiency of the regeneration allowed solid supports with streptavidin to be used many times, here exemplified with the multiple re-use of streptavidin beads used for sample preparation prior to automated DNA sequencing. The results suggest that streptavidin regeneration can be introduced as an improvement in existing methods and assays based on the streptavidin system as well as emerging solid phase applications in fields, such as microfluiclics and nanotechnology.

  • 144. Hu, M.
    et al.
    Ayub, Q.
    Guerra-Assunção, J. A.
    Long, Q.
    Ning, Z.
    Huang, N.
    Romero, I. G.
    Mamanova, L.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Liu, X.
    Coffey, A. J.
    Turner, D. J.
    Swerdlow, H.
    Burton, J.
    Quail, M. A.
    Conrad, D. F.
    Enright, A. J.
    Tyler-Smith, C.
    Xue, Y.
    Exploration of signals of positive selection derived from genotype-based human genome scans using re-sequencing data2012In: Human Genetics, ISSN 0340-6717, E-ISSN 1432-1203, Vol. 131, no 5, p. 665-674Article in journal (Refereed)
    Abstract [en]

    We have investigated whether regions of the genome showing signs of positive selection in scans based on haplotype structure also show evidence of positive selection when sequence-based tests are applied, whether the target of selection can be localized more precisely, and whether such extra evidence can lead to increased biological insights. We used two tools: simulations under neutrality or selection, and experimental investigation of two regions identified by the HapMap2 project as putatively selected in human populations. Simulations suggested that neutral and selected regions should be readily distinguished and that it should be possible to localize the selected variant to within 40 kb at least half of the time. Re-sequencing of two ∼300 kb regions (chr4:158Mb and chr10:22Mb) lacking known targets of selection in HapMap CHB individuals provided strong evidence for positive selection within each and suggested the micro-RNA gene hsa-miR-548c as the best candidate target in one region, and changes in regulation of the sperm protein gene SPAG6 in the other.

  • 145. Hu, Y.
    et al.
    Zhu, Z.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers University of Technology, Sweden.
    Siewers, V.
    Heterologous transporter expression for improved fatty alcohol secretion in yeast2018In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 45, p. 51-58Article in journal (Refereed)
    Abstract [en]

    The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known as a free fatty acid importer to date. We furthermore successfully identified the functional domain of FATP1 involved in fatty alcohol export through domain exchange between FATP1 and another transporter, FATP4. This study may facilitate a successful commercialization of fatty alcohol production in yeast and inspire the design of novel cell factories.

  • 146.
    Hu, Yue
    et al.
    KTH, School of Biotechnology (BIO).
    Ndegwa, Nelson
    Karolinska Institutet, Department of Medical Epidemiology and Biostatistics (MEB).
    Alneberg, Johannes
    KTH, School of Biotechnology (BIO).
    Johansson, Sebastian
    KTH, School of Biotechnology (BIO).
    Logue, Jürg
    Linnaeus University, Centre for Ecology and Evolution in Microbial Model Systems.
    Huss, Mikael
    Stockholm University.
    Käller, Max
    KTH, School of Biotechnology (BIO).
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fagerberg, Jens
    Stockholm Vatten och Avfall AB.
    Andersson, Anders
    KTH, School of Biotechnology (BIO).
    Stationary and portable sequencing-based approaches for tracing wastewater contamination in urban stormwater systemsManuscript (preprint) (Other academic)
    Abstract [en]

    Urban sewer systems consist of wastewater and stormwater sewers, of which typically only

    the wastewater is processed before being discharged. Occasionally, misconnections or

    damages in the network occur, resulting in wastewater entering the stormwater system and

    being discharged without prior processing. Cultivation of faecal indicator bacteria, such as

    Escherichia coli (E. coli), is the current standard for tracing wastewater contamination. This

    method is cheap but cannot be employed in the field and is characterised by its limited

    specificity. Here, we compared the E. coli culturing approach with two different DNA

    sequencing-based methodologies (i.e., 16S rRNA amplicon sequencing on the Illumina

    MiSeq platform and shotgun metagenomic sequencing on an Oxford Nanopore MinIOn

    device), analysing 73 stormwater samples collected throughout the Stockholm city areas.

    High correlations were obtained between E. coli culturing counts and frequencies of human

    gut microbiome sequencing reads (via amplicon sequencing), indicating that E. coli is indeed

    a good indicator of faecal contamination. In contrast to E.coli culturing, amplicon sequencing

    could, however, further distinguish between two different sources of contamination in an

    area, where misconnections in the stormwater system were later on detected. Shotgun

    metagenomic sequencing on a subset of the samples using the portable Oxford Nanopore

    MinION real-time sequencing device correlated well with the amplicon sequencing data. In

    summary, this study shows that DNA sequencing allows distinguishing different

    contamination sources in stormwater systems and demonstrates the potential of using a

    portable sequencing device in the field for tracking faecal contamination.

  • 147.
    Hu, Yue O. O.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Karlson, Bengt
    Charvet, Sophie
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Diversity of Pico- to Mesoplankton along the 2000 km Salinity Gradient of the Baltic Sea2016In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 7, article id 679Article in journal (Refereed)
    Abstract [en]

    Microbial plankton form the productive base of both marine and freshwater ecosystems and are key drivers of global biogeochemical cycles of carbon and nutrients. Plankton diversity is immense with representations from all major phyla within the three domains of life. So far, plankton monitoring has mainly been based on microscopic identification, which has limited sensitivity and reproducibility, not least because of the numerical majority of plankton being unidentifiable under the light microscope. High-throughput sequencing of taxonomic marker genes offers a means to identify taxa inaccessible by traditional methods; thus, recent studies have unveiled an extensive previously unknown diversity of plankton. Here, we conducted ultra-deep Illumina sequencing (average 105 sequences/sample) of rRNA gene amplicons of surface water eukaryotic and bacterial plankton communities sampled in summer along a 2000 km transect following the salinity gradient of the Baltic Sea. Community composition was strongly correlated with salinity for both bacterial and eukaryotic plankton assemblages, highlighting the importance of salinity for structuring the biodiversity within this ecosystem. In contrast, no clear trends in alpha-diversity for bacterial or eukaryotic communities could be detected along the transect. The distribution of major planktonic taxa followed expected patterns as observed in monitoring programs, but groups novel to the Baltic Sea were also identified, such as relatives to the coccolithophore Erniliana huxleyi detected in the northern Baltic Sea. This study provides the first ultra-deep sequencing-based survey on eukaryotic and bacterial plankton biogeography in the Baltic Sea.

  • 148.
    Hugerth, Luisa
    KTH, School of Biotechnology (BIO), Gene Technology. Science for Life Laboratory.
    High-throughput DNA Sequencingin Microbial Ecology: Methods and Applications2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Microorganisms play central roles in planet Earth’s geochemical cycles, in food production, and in health and disease of humans and livestock. In spite of this, most microbial life forms remain unknown and unnamed, their ecological importance and potential technological applications beyond the realm of speculation. This is due both to the magnitude of microbial diversity and to technological limitations. Of the many advances that have enabled microbiology to reach new depth and breadth in the past decade, one of the most important is affordable high-throughput DNA sequencing. This technology plays a central role in each paper in this thesis.

    Papers I and II are focused on developing methods to survey microbial diversity based on marker gene amplification and sequencing. In Paper I we proposed a computational strategy to design primers with the highest coverage among a given set of sequences and applied it to drastically improve one of the most commonly used primer pairs for ecological surveys of prokaryotes. In Paper II this strategy was applied to an eukaryotic marker gene. Despite their importance in the food chain, eukaryotic microbes are much more seldom surveyed than bacteria. Paper II aimed at making this domain of life more amenable to high-throughput surveys.

    In Paper III, the primers designed in papers I and II were applied to water samples collected up to twice weekly from 2011 to 2013 at an offshore station in the Baltic proper, the Linnaeus Microbial Observatory. In addition to tracking microbial communities over these three years, we created predictive models for hundreds of microbial populations, based on their co-occurrence with other populations and environmental factors.

    In paper IV we explored the entire metagenomic diversity in the Linnaeus Microbial Observatory. We used computational tools developed in our group to construct draft genomes of abundant bacteria and archaea and described their phylogeny, seasonal dynamics and potential physiology. We were also able to establish that, rather than being a mixture of genomes from fresh and saline water, the Baltic Sea plankton community is composed of brackish specialists which diverged from other aquatic microorganisms thousands of years before the formation of the Baltic itself.

  • 149.
    Hugerth, Luisa
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. Science for Life Laboratory.
    Lindh, Markus
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Sjöqvist, Conny
    KTH, School of Biotechnology (BIO), Gene Technology.
    Carina, Bunse
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Legrand, Catherine
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Pinhassi, Jarone
    Centre for Ecology and Evolution in Microbial model Systems - Linnaeus University.
    Andersson, Anders
    KTH, School of Biotechnology (BIO), Gene Technology.
    Seasonal dynamics and interactions among Baltic Sea prokaryoticand eukaryotic plankton assemblagesManuscript (preprint) (Other academic)
    Abstract [en]

    One of the main goals of microbial ecology is to identify the mechanismsthat regulate patterns in community structure at temporal scalescompatible with populations’ turnover times across complete seasonalcycles. Here, we examined high-frequency temporal dynamics of marineplankton from a sampling effort covering 2011-2013, roughly twice weekly,comprising 144 samples. Bacterial and eukaryotic communities wereprofiled by 16S and 18S high-throughput sequencing, respectively.Interestingly, we found that no combination of the measured environmentalparameters could predict a significant proportion of the variation inpopulation dynamics of bacterioplankton, and even less so for eukaryoticplankton. Large differences in physicochemical conditions and communitycomposition typical of temperate climates mean that different regimes canquickly succeed each other over the year, with the relative importance ofdifferent drivers changing equally rapidly. Nevertheless, our approachrevealed interesting recurrent co-occurrence patterns across distinctenvironmental changes. Hence, we could make abundance predictions formore than half of the most frequent OTUs based on interactions with otherOTUs. These results suggests that a complex set of biotic interactions arecontributing to temporal patterns among planktonic assemblages despiterapid changes in environmental conditions.

  • 150.
    Hugerth, Luisa W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Analysing Microbial Community Composition through Amplicon Sequencing: From Sampling to Hypothesis Testing2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 1561Article, review/survey (Refereed)
    Abstract [en]

    Microbial ecology as a scientific field is fundamentally driven by technological advance. The past decade's revolution in DNA sequencing cost and throughput has made it possible for most research groups to map microbial community composition in environments of interest. However, the computational and statistical methodology required to analyse this kind of data is often not part of the biologist training. In this review, we give a historical perspective on the use of sequencing data inmicrobial ecology and restate the current need for this method; but also highlight the major caveats with standard practices for handling these data, from sample collection and library preparation to statistical analysis. Further, we outline the main new analytical tools that have been developed in the past few years to bypass these caveats, as well as highlight the major requirements of common statistical practices and the extent to which they are applicable to microbial data. Besides delving into the meaning of select alpha- and beta-diversity measures, we give special consideration to techniques for finding the main drivers of community dissimilarity and for interaction network construction. While every project design has specific needs, this review should serve as a starting point for considering what options are available.

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