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  • 1.
    Ali, Raja Hashim
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Khan, Ammad Aslam
    Tracing the evolution of FERM domain of Kindlins2014In: Molecular Phylogenetics and Evolution, ISSN 1055-7903, E-ISSN 1095-9513, Vol. 80, p. 193-204Article in journal (Refereed)
    Abstract [en]

    Kindlin proteins represent a novel family of evolutionarily conserved FERM domain containing proteins (FDCPs) and are members of B4.1 superfamily. Kindlins consist of three conserved protein homologs in vertebrates: Kindlin-1, Kindlin-2 and Kindlin-3. All three homologs are associated with focal adhesions and are involved in Integrin activation. FERM domain of each Kindlin is bipartite and plays a key role in Integrin activation. A single ancestral Kindlin protein can be traced back to earliest metazoans, e.g., to Parazoa. This protein underwent multiple rounds of duplication in vertebrates, leading to the present Kindlin family. In this study, we trace phylogenetic and evolutionary history of Kindlin FERM domain with respect to FERM domain of other FDCPs. We show that FERM domain in Kindlin homologs is conserved among Kindlins but amount of conservation is less in comparison with FERM domain of other members in B4.1 superfamily. Furthermore, insertion of Pleckstrin Homology like domain in Kindlin FERM domain has important evolutionary and functional consequences. Important residues in Kindlins are traced and ranked according to their evolutionary significance. The structural and functional significance of high ranked residues is highlighted and validated by their known involvement in Kindlin associated diseases. In light of these findings, we hypothesize that FERM domain originated from a proto-Talin protein in unicellular or proto-multicellular organism and advent of multi-cellularity was accompanied by burst of FDCPs, which supported multi-cellularity functions required for complex organisms. This study helps in developing a better understanding of evolutionary history of FERM domain of FDCPs and the role of FERM domain in metazoan evolution.

  • 2.
    Ardalan, Arman
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Molecular Profiling of the Population Dynamics: Foundation and Expansion of an Archaic Domesticate2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    "An ‘exponential growth of science’ throughout modern history has been frequently boasted by numerous narcissistic accounts of ‘modern humanity.’ Nonetheless, ‘modern science’ seems to have overwhelmingly compromised on its original promises by fitting into an ‘industrial scheme.’ With this concern, ‘molecular phylogeographics with conservational ambitions’ would look an intact ground for research efforts in a ‘school of biotechnology.’ The dog (Canis familiaris) as an earliest domestic animal has a history of conflicts over its origins and dispersal. Having those disputes addressed, valuable knowledge could be acquired on the nature and dynamics of domestication, and of human societies particularly of pre-agricultural ages. We employed two most widely-used genealogical markers, the mitochondrial DNA (mtDNA) and the non-recombining portion of the Y-chromosome (NRY), to address dog demography. Through 582 bps of mtDNA Control Region, complemented with whole mitochondrial genomes, it was established that almost all maternal lineages of the domestic dog worldwide coalesce to a population of at least 51 and perhaps many more female wolves in Asia South of Yangtze River (ASY) approximately 16,000 years before present (BP). This was based on the presence of a maximal diversity in this area, a descending gradient of diversity outward it, and a ubiquitous population structure everywhere in the world. A closer examination of this portrait in Southwest Asia (SwAsia) and the Fertile Crescent (FC), a region which has supplied persuasive evidence on early presence of the domestic dog, retrieved the same information, with implications for backbreeding with the local wolf population. Meanwhile, analyses of mtDNA dispersal showed that dogs took the long way via land to Madagascar Island, and not together with humans via sea. By the other approach, the NRY data in 14,437 bps length supplemented the mtDNA in reporting the height of diversity from ASY with a founding population of at least 13 male wolves, but expectably produced lower inter-regional differentiation by diversity. Screening of NRY by a SNP assay in the dingoes of Australia Island as a population of feral dogs revealed restricted and similar dispersal patterns for sires and dams. Prospects of ancient, multilocus and whole genome assays with the emerging high-throughput technologies has still more to promise on finer elaborations of these issues."

  • 3.
    Asp, Michaela
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Salmen, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Stahl, Patrik L.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Felldin, Ulrika
    Lofling, Marie
    Navarro, Jose Fernandez
    Maaskola, Jonas
    Eriksson, Maria J.
    Persson, Bengt
    Corbascio, Matthias
    Persson, Hans
    Linde, Cecilia
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Spatial detection of fetal marker genes expressed at low level in adult human heart tissue2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 12941Article in journal (Refereed)
    Abstract [en]

    Heart failure is a major health problem linked to poor quality of life and high mortality rates. Hence, novel biomarkers, such as fetal marker genes with low expression levels, could potentially differentiate disease states in order to improve therapy. In many studies on heart failure, cardiac biopsies have been analyzed as uniform pieces of tissue with bulk techniques, but this homogenization approach can mask medically relevant phenotypes occurring only in isolated parts of the tissue. This study examines such spatial variations within and between regions of cardiac biopsies. In contrast to standard RNA sequencing, this approach provides a spatially resolved transcriptome- and tissue-wide perspective of the adult human heart, and enables detection of fetal marker genes expressed by minor subpopulations of cells within the tissue. Analysis of patients with heart failure, with preserved ejection fraction, demonstrated spatially divergent expression of fetal genes in cardiac biopsies.

  • 4.
    Aurell, Erik
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST). Aalto University, Finland.
    Innocenti, Nicolas
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST). The Hebrew University of Jerusalem, Israel.
    Zhou, Hai-Jun
    State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Sciences, Beijing 100190, China.
    The bulk and the tail of minimal absent words in genome sequences2016In: Physical Biology, ISSN 1478-3967, E-ISSN 1478-3975, Vol. 13, no 2, article id 026004Article in journal (Refereed)
    Abstract [en]

    Minimal absent words (MAW) of a genomic sequence are subsequences that are absent themselves but the subwords of which are all present in the sequence. The characteristic distribution of genomic MAWs as a function of their length has been observed to be qualitatively similar for all living organisms, the bulk being rather short, and only relatively few being long. It has been an open issue whether the reason behind this phenomenon is statistical or reflects a biological mechanism, and what biological information is contained in absent words. % In this work we demonstrate that the bulk can be described by a probabilistic model of sampling words from random sequences, while the tail of long MAWs is of biological origin. We introduce the novel concept of a core of a minimal absent word, which are sequences present in the genome and closest to a given MAW. We show that in bacteria and yeast the cores of the longest MAWs, which exist in two or more copies, are located in highly conserved regions the most prominent example being ribosomal RNAs (rRNAs). We also show that while the distribution of the cores of long MAWs is roughly uniform over these genomes on a coarse-grained level, on a more detailed level it is strongly enhanced in 3' untranslated regions (UTRs) and, to a lesser extent, also in 5' UTRs. This indicates that MAWs and associated MAW cores correspond to fine-tuned evolutionary relationships, and suggest that they can be more widely used as markers for genomic complexity.

  • 5. Bergquist, Helen
    et al.
    Rocha, Cristina S. J.
    Alvarez-Asencio, Ruben
    KTH, School of Chemical Science and Engineering (CHE), Chemistry. Universitario de Cantoblanco, Spain.
    Nguyen, Chi-Hung
    Rutland, Mark W.
    KTH, School of Chemical Science and Engineering (CHE), Chemistry.
    Smith, C. I. Edvard
    Good, Liam
    Nielsen, Peter E.
    Zain, Rula
    Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)(n) Repeats by PNA or LNA Targeting2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 11, article id e0165788Article in journal (Refereed)
    Abstract [en]

    Expansion of (GAA)(n) repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich's ataxia. (GAA)(n) expansions form non-canonical structures, including intramolecular triplex (H-DNA), and Rloops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)(n) expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)(n) repeats of different lengths (short: n= 9, medium: n= 75 or long: n= 115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA) 4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT) 5-PNA did not. We present evidence that (GAA) 4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT) 5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.

  • 6. Bjursell, Magnus K.
    et al.
    Blom, Henk J.
    Cayuela, Jordi Asin
    Engvall, Martin L.
    Lesko, Nicole
    Balasubramaniam, Shanti
    Brandberg, Goran
    Halldin, Maria
    Falkenberg, Maria
    Jakobs, Cornelis
    Smith, Desiree
    Struys, Eduard
    von Dobeln, Ulrika
    Gustafsson, Claes M.
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Wedell, Anna
    Adenosine Kinase Deficiency Disrupts the Methionine Cycle and Causes Hypermethioninemia, Encephalopathy, and Abnormal Liver Function2011In: American Journal of Human Genetics, ISSN 0002-9297, E-ISSN 1537-6605, Vol. 89, no 4, p. 507-515Article in journal (Refereed)
    Abstract [en]

    Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (Ado Met), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism.

  • 7.
    Blomstergren, Anna
    et al.
    KTH, Superseded Departments, Biotechnology.
    Lundin, A.
    Nilsson, C.
    Engstrand, L.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Comparative analysis of the complete cag pathogenicity island sequence in four Helicobacter pylori isolates2004In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 328, p. 85-93Article in journal (Refereed)
    Abstract [en]

    The cytotoxin-associated gene (cag) pathogenicity island (PAI) is important for the virulence of Helicobacter pylori. In this study, we have compared the complete nucleotide sequence of the cag PAI in four clinical isolates. These isolates were selected from patients matched for age and sex from the same geographical region. The patients suffered from either gastric cancer (Ca52 and Ca73) or duodenal ulcer (Du23:2 and Du52:2). All four strains induced an interleukin (IL)-8 response in AGS cells and translocated CagA into host cells where the protein was tyrosine phosphorylated, and thus harboured a functional type W secretion system encoded by the cag PAL The cagA gene contains a variable region close to its 3' end. Different compositions of this region has been proposed to exert various degrees of morphological changes in cultured gastric epithelial cells, and there are indications that the structure of the repetitive region is connected to the severity of disease. One of the studied strains (Du23:2) possessed five Westem-type repeat regions while the other three strains harboured one Western-type repeat. Strain Du23:2 also contained a major rearrangement or large insertion/ duplication in the intergenic region between HP0546 and HP0547 (cagA). Sequence similar to that of genes HP0510 and HP0509 was found in the 5' end of this region. The 3' end was-similar to the corresponding region of strain ATCC 43504, including a mini IS605 element and a duplication of the 3' end of the cag PAL Finally, a novel gene was identified in the cag PAI in three of the sequenced strains at the position of HP0521. This gene, HP0521B, is present in approximately half of Swedish H. pylori isolates.

  • 8. Corander, Jukka
    et al.
    Gyllenberg, Mats
    Koski, Timo
    KTH, School of Engineering Sciences (SCI), Mathematics (Dept.), Mathematical Statistics.
    Learning Genetic Population Structures Using Minimization of Stochastic Complexity2010In: Entropy, ISSN 1099-4300, E-ISSN 1099-4300, Vol. 12, no 5, p. 1102-1124Article in journal (Refereed)
    Abstract [en]

    Considerable research efforts have been devoted to probabilistic modeling of genetic population structures within the past decade. In particular, a wide spectrum of Bayesian models have been proposed for unlinked molecular marker data from diploid organisms. Here we derive a theoretical framework for learning genetic population structure of a haploid organism from bi-allelic markers for which potential patterns of dependence are a priori unknown and to be explicitly incorporated in the model. Our framework is based on the principle of minimizing stochastic complexity of an unsupervised classification under tree augmented factorization of the predictive data distribution. We discuss a fast implementation of the learning framework using deterministic algorithms.

  • 9. Costa, H.
    et al.
    Xu, X.
    Overbeek, G.
    Vasaikar, S.
    Pawan K. Patro, C.
    Kostopoulou, O. N.
    Jung, M.
    Shafi, G.
    Ananthaseshan, S.
    Tsipras, G.
    Davoudi, B.
    Mohammad, A. -A
    Lam, H.
    Strååt, Klas
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sweden.
    Wilhelmi, V.
    Shang, M.
    Tegner, J.
    Tong, J. C.
    Wong, K. T.
    Söderberg-Naucler, C.
    Yaiw, K. -C
    Human cytomegalovirus may promote tumour progression by upregulating arginase-22016In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 7, no 30, p. 47221-47231Article in journal (Refereed)
    Abstract [en]

    Background: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. Results: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. Methods: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2- overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). Conclusions: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.

  • 10. Eisfeldt, Jesper
    et al.
    Nazaryan-Petersen, Lusine
    Lundin, Johanna Lundin
    Pettersson, Maria
    Nilsson, Daniel
    Wincent, Josephine
    Lieden, Agne
    Vezzi, Francesco
    Wirta, Valteri
    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.
    Duelund, Tina
    Houssari, Rayan
    Pignata, Laura
    Bak, Mads
    Tommerup, Niels
    Lundberg, Elisabeth Syk
    Tumer, Zeynep
    Lindstrand, Anna
    Whole genome characterization of array defined clustered CNVs reveals two distinct complex rearrangement subclasses generated through either non homologous repair or template switching2017In: Molecular Cytogenetics, ISSN 1755-8166, E-ISSN 1755-8166, Vol. 10Article in journal (Other academic)
  • 11. Engstrom, Karin
    et al.
    Wojdacz, Tomasz K.
    Marabita, Francesco
    Ewels, Philip
    Käller, Max
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vezzi, Francesco
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Prezza, Nicola
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gruselius, Joel
    Vahter, Marie
    Broberg, Karin
    Transcriptomics and methylomics of CD4-positive T cells in arsenic-exposed women2017In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 91, no 5, p. 2067-2078Article in journal (Refereed)
    Abstract [en]

    Arsenic, a carcinogen with immunotoxic effects, is a common contaminant of drinking water and certain food worldwide. We hypothesized that chronic arsenic exposure alters gene expression, potentially by altering DNA methylation of genes encoding central components of the immune system. We therefore analyzed the transcriptomes (by RNA sequencing) and methylomes (by target-enrichment next-generation sequencing) of primary CD4-positive T cells from matched groups of four women each in the Argentinean Andes, with fivefold differences in urinary arsenic concentrations (median concentrations of urinary arsenic in the lower- and high-arsenic groups: 65 and 276 mu g/l, respectively). Arsenic exposure was associated with genome-wide alterations of gene expression; principal component analysis indicated that the exposure explained 53% of the variance in gene expression among the top variable genes and 19% of 28,351 genes were differentially expressed (false discovery rate < 0.05) between the exposure groups. Key genes regulating the immune system, such as tumor necrosis factor alpha and interferon gamma, as well as genes related to the NF-kappa-beta complex, were significantly downregulated in the high-arsenic group. Arsenic exposure was associated with genome-wide DNA methylation; the high-arsenic group had 3% points higher genome-wide full methylation (> 80% methylation) than the lower-arsenic group. Differentially methylated regions that were hyper-methylated in the high-arsenic group showed enrichment for immune-related gene ontologies that constitute the basic functions of CD4-positive T cells, such as isotype switching and lymphocyte activation and differentiation. In conclusion, chronic arsenic exposure from drinking water was related to changes in the transcriptome and methylome of CD4-positive T cells, both genome wide and in specific genes, supporting the hypothesis that arsenic causes immunotoxicity by interfering with gene expression and regulation.

  • 12. 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.

  • 13. Gallus, S.
    et al.
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Senckenberg Gesellschaft für Naturforschung, Germany .
    Kumar, V.
    Dodt, W. G.
    Janke, A.
    Schumann, G. G.
    Nilsson, M. A.
    Evolutionary histories of transposable elements in the genome of the largest living marsupial carnivore, the tasmanian devil2015In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 32, no 5, p. 1268-1283Article in journal (Refereed)
    Abstract [en]

    The largest living carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii), is the sole survivor of a lineage originating about 12 Ma. We set out to investigate the spectrum of transposable elements found in the Tasmanian devil genome, the first high-coverage genome of an Australian marsupial. Marsupial genomes have been shown to have the highest amount of transposable elements among vertebrates. We analyzed the horizontally transmitted DNA transposons OC1 and hAT-1-MEu in the Tasmanian devil genome. OC1 is present in all carnivorous marsupials, while having a very limited distribution among the remaining Australian marsupial orders. In contrast, hAT-1-MEu is present in all Australian marsupial orders, and has so far only been identified in a few placental mammals. We screened 158 introns for phylogenetically informative retrotransposons in the order Dasyuromorphia, and found that the youngest SINE (Short INterspersed Element), WSINE1, is no longer active in the subfamily Dasyuridae. The lack of detectable WSINE1 activity in this group may be due to a retrotransposon inactivation event approximately 30 Ma. We found that the Tasmanian devil genome contains a relatively low number of continuous full-length LINE-1 (Long INterspersed Element 1, L1) retrotransposons compared with the opossum genome. Furthermore, all L1 elements in the Tasmanian devil appeared to be nonfunctional. Hidden Markov Model approaches suggested that other potential sources of functional reverse transcriptase are absent from the genome. We discuss the issues associated with assembling long, highly similar L1 copies from short read Illumina data and describe how assembly artifacts can potentially lead to erroneous conclusions.

  • 14. Gambelunghe, G.
    et al.
    Ghaderi, M.
    Gharizadeh, Baback
    KTH, Superseded Departments, Biotechnology.
    Brozzetti, A.
    Tortoioli, C.
    Del Sindaco, P.
    Sanjeevi, C. B.
    Hjelmstrom, P.
    Sirsjo, A.
    Nyrén, Pål
    KTH, Superseded Departments, Biochemistry and Biotechnology.
    Santeusanio, F.
    Falorni, A.
    Lack of association of human chemokine receptor gene polymorphisms CCR2-64I and CCR5-Delta 32 with autoimmune Addison's disease2004In: European journal of immunogenetics, ISSN 0960-7420, E-ISSN 1365-2370, Vol. 31, no 2, p. 73-76Article in journal (Refereed)
    Abstract [en]

    The attraction of leukocytes to tissues is essential for inflammation and the initiation of the autoimmune reaction. The process is controlled by chemokines, which are chemotactic cytokines. We investigated whether human chemokine receptor gene polymorphisms, namely CCR5-Delta32 and CCR2-64I, are associated with susceptibility to autoimmune Addison's disease. Genotyping was performed in 56 patients and 127 healthy controls by a new method using pyrosequencing for CCR2-64I and by polymerase chain reaction and detecting gel for CCR5-Delta32. None of the CCR2 or CCR5 alleles was found to be associated, either positively or negatively, with disease risk. Our results indicate that the CCR2-64I and CCR5-Delta32 gene polymorphisms do not play a major role in conferring genetic risk for, and/or protection against, autoimmune Addison's disease.

  • 15. Gottlieb, Bruce
    et al.
    Alvarado, Carlos
    Wang, Chunlin
    Gharizadeh, Baback
    Babrzadeh, Farbod
    Stanford Genome Technology Center, Stanford University, Palo Alto, CA, United States .
    Richards, Brent
    Batist, Gerald
    Basik, Mark
    Beitel, Lenore K.
    Trifiro, Mark
    Making Sense of Intratumor Genetic Heterogeneity: Altered Frequency of Androgen Receptor CAG Repeat Length Variants in Breast Cancer Tissues2013In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 34, no 4, p. 610-618Article in journal (Refereed)
    Abstract [en]

    To examine the significance of intratumor genetic heterogeneity (ITGH) of the androgen receptor (AR) gene in breast cancer, patient-matched samples of laser capture microdissected breast tumor cells, adjacent normal breast epithelia cells, and peripheral blood leukocytes were sequenced using a novel next generation sequencing protocol. This protocol measured the frequency of distribution of a variable AR CAG repeat length, a functional polymorphism associated with breast cancer risk. All samples exhibited some degree of ITGH with up to 30 CAG repeat length variants identified. Each type of tissue exhibited a different distribution profile of CAG repeat lengths with substantial differences in the frequencies of zero and 1825 CAG AR variants. Tissue differences in the frequency of ARs with each of these CAG repeat lengths were significant as measured by paired, twin t-tests. These results suggest that preferential selection of 1825 CAG repeat length variants in breast tumors may be associated with breast cancer, and support the observation that shorter CAG repeats may protect against breast cancer. They also suggest that merely identifying variant genes will be insufficient to determine the critical mutational events of oncogenesis, which will require measuring the frequency of distribution of mutations within cancerous and matching normal tissues.

  • 16. 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.

  • 17.
    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.

  • 18.
    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.

  • 19. 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.

  • 20. 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.

  • 21.
    Hugerth, Luisa W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Larsson, John
    Alneberg, Johannes
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lindh, Markus V.
    Legrand, Catherine
    Pinhassi, Jarone
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Metagenome-assembled genomes uncover a global brackish microbiome2015In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 16, article id 279Article in journal (Refereed)
    Abstract [en]

    Background: Microbes are main drivers of biogeochemical cycles in oceans and lakes. Although the genome is a foundation for understanding the metabolism, ecology and evolution of an organism, few bacterioplankton genomes have been sequenced, partly due to difficulties in cultivating them. Results: We use automatic binning to reconstruct a large number of bacterioplankton genomes from a metagenomic time-series from the Baltic Sea, one of world's largest brackish water bodies. These genomes represent novel species within typical freshwater and marine clades, including clades not previously sequenced. The genomes' seasonal dynamics follow phylogenetic patterns, but with fine-grained lineage-specific variations, reflected in gene-content. Signs of streamlining are evident in most genomes, and estimated genome sizes correlate with abundance variation across filter size fractions. Comparing the genomes with globally distributed metagenomes reveals significant fragment recruitment at high sequence identity from brackish waters in North America, but little from lakes or oceans. This suggests the existence of a global brackish metacommunity whose populations diverged from freshwater and marine relatives over 100,000 years ago, long before the Baltic Sea was formed (8000 years ago). This markedly contrasts to most Baltic Sea multicellular organisms, which are locally adapted populations of freshwater or marine counterparts. Conclusions: We describe the gene content, temporal dynamics and biogeography of a large set of new bacterioplankton genomes assembled from metagenomes. We propose that brackish environments exert such strong selection that lineages adapted to them flourish globally with limited influence from surrounding aquatic communities.

  • 22.
    Innocenti, Nicolas
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Data Analysis and Next Generation Sequencing : Applications in Microbiology.2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Next Generation Sequencing (NGS) is a new technology that has revolutionized the way we study living organisms. Where previously only a few genes could be studied at a time through targeted direct probing, NGS offers the possibility to perform measurements for a whole genome at once. The drawback is that the amount of data generated in the process is large and extracting useful information from it requires new methods to process and analyze it.

    The main contribution of this thesis is the development of a novel experimental method coined tagRNA-seq, combining 5’tagRACE, a previously developed technique, with RNA-sequencing technology. Briefly, tagRNA-seq makes it possible to identify the 5’ ends of RNAs in bacteria and directly probe for their type, primary or processed, by ligating short RNA sequences, the tags, to the beginnings of RNA molecules. We used the method to directly probe for transcription start and processing sites in two bacterial species, Escherichiacoli and Enterococcus faecalis. It was also used to study polyadenylation in E. coli, where the ability to identify processed RNA molecules proved to be useful to separate direct and indirect regulatory effects of this mechanism. We also demonstrate how data from tagRNA-seq experiments can be used to increase confidence on the discovery of anti-sense transcripts in bacteria. Analyses of RNA-seq data obtained in the context of these experiments revealed subtle artifacts in the coverage signal towards gene ends, that we were able to explain and quantify based Kolmogorov’s broken stick model. We also discovered evidences for circularization of a few RNA transcripts, both in our own data sets and publicly available data.

    Designing the tags used in tagRNA-seq led us to the problem of words absent from a text. We focus on a particular subset of these, the minimal absent words (MAWs), and develop a theory providing a complete description of their size distribution in random text. We also show that MAWs in genomes from viruses and living organisms almost always exhibit a behavior different from random texts in the tail of the distribution, and that MAWs from this tail are closely related to sequences present in the genome that preferentially appear in regions with important regulatory functions.

    Finally, and independently from tagRNA-seq, we propose a new approach to the problem of bacterial community reconstruction in metagenomic, based on techniques from compressed sensing. We provide a novel algorithm competing with state-of-the-art techniques in the field.

  • 23.
    Innocenti, Nicolas
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Golumbeanu, Monica
    Department of Biosystems Science and Engineering, ETH Zürich, CH-4058, Basel, Switzerland.
    Fouquier d'Hérouël, Aymeric
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. Luxembourg Centre for Systems Biomedicine, University of Luxembourg, L-4362, Esch-sur-Alzette, Luxembourg.
    Lacoux, Caroline
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Bonnin, Rémy A.
    Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS, UMR8621, F-91405, Orsay, France.
    Kennedy, Sean P.
    INRA, MetaGenoPolis US1367, Domaine de Vilvert, F-78350, Jouy-en-Josas, France.
    Wessner, Francoise
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Serror, Pascale
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Bouloc, Philippe
    Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS, UMR8621, F-91405, Orsay, France.
    Repoila, Francis
    INRA, UMR1319 Micalis, Domaine de Vilvert, F-78352, Jouy-en-Josas, France.
    Aurell, Erik
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB.
    Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis2015In: RNA, ISSN 1355-8382Article in journal (Refereed)
    Abstract [en]

    Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5' RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA-and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small-and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.

  • 24. Jiang, Rays H. Y.
    et al.
    de Bruijn, Irene
    Haas, Brian J.
    Belmonte, Rodrigo
    Loebach, Lars
    Christie, James
    van den Ackerveken, Guido
    Bottin, Arnaud
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Diaz-Moreno, Sara M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Dumas, Bernard
    Fan, Lin
    Gaulin, Elodie
    Govers, Francine
    Grenville-Briggs, Laura J.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Horner, Neil R.
    Levin, Joshua Z.
    Mammella, Marco
    Meijer, Harold J. G.
    Morris, Paul
    Nusbaum, Chad
    Oome, Stan
    Phillips, Andrew J.
    van Rooyen, David
    Rzeszutek, Elzbieta
    KTH, School of Biotechnology (BIO), Glycoscience.
    Saraiva, Marcia
    Secombes, Chris J.
    Seidl, Michael F.
    Snel, Berend
    Stassen, Joost H. M.
    Sykes, Sean
    Tripathy, Sucheta
    van den Berg, Herbert
    Vega-Arreguin, Julio C.
    Wawra, Stephan
    Young, Sarah K.
    Zeng, Qiandong
    Dieguez-Uribeondo, Javier
    Russ, Carsten
    Tyler, Brett M.
    van West, Pieter
    Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica2013In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 9, no 6, p. e1003272-Article in journal (Refereed)
    Abstract [en]

    Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.

  • 25. Ka, S.
    et al.
    Fitzsimmons, C.
    Jacobsson, L.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Siegel, P. B.
    Andersson, L.
    Hallböök, F.
    Expression analysis of growth and energy regulation-associated genes in two divergent chicken strains2005In: Trends in Comparative Endocrinology and Neurobiology, New York Academy of Sciences, 2005, p. 357-359Conference paper (Refereed)
    Abstract [en]

    We have studied differential expression of genes using cDNA arrays in the hypothalamic region of two divergent chicken lines, which differ in body weight and feeding behavior. Several transcripts from genes in metabolic networks as well as from retroviruses were differentially expressed.

  • 26.
    Kaewthai, Nomchit
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gendre, Delphine
    Eklöf, Jens M.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Ibatullin, Farid M.
    Ezcurra, Ines
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bhalerao, Rishikesh P.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience.
    Group III-A XTH genes encode predominant xyloglucan endo hydrolase active in expanding tissues of Arabidopsis thalianaManuscript (preprint) (Other academic)
  • 27.
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    On Transcriptome Sequencing2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.

  • 28.
    Klütsch, Cornelya
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Seppälä, E. H.
    Fall, T.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Gene Technology.
    Hedhammar, Å.
    Lohi, H.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia2011In: Animal Genetics, ISSN 0268-9146, E-ISSN 1365-2052, Vol. 42, no 1, p. 100-103Article in journal (Refereed)
    Abstract [en]

    P>The domestic dog mitochondrial DNA (mtDNA)-gene pool consists of a homogenous mix of haplogroups shared among all populations worldwide, indicating that the dog originated at a single time and place. However, one small haplogroup, subclade d1, found among North Scandinavian/Finnish spitz breeds at frequencies above 30%, has a clearly separate origin. We studied the genetic and geographical diversity for this phylogenetic group to investigate where and when it originated and whether through independent domestication of wolf or dog-wolf crossbreeding. We analysed 582 bp of the mtDNA control region for 514 dogs of breeds earlier shown to harbour d1 and possibly related northern spitz breeds. Subclade d1 occurred almost exclusively among Swedish/Finnish Sami reindeer-herding spitzes and some Swedish/Norwegian hunting spitzes, at a frequency of mostly 60-100%. Genetic diversity was low, with only four haplotypes: a central, most frequent, one surrounded by two haplotypes differing by an indel and one differing by a substitution. The substitution was found in a single lineage, as a heteroplasmic mix with the central haplotype. The data indicate that subclade d1 originated in northern Scandinavia, at most 480-3000 years ago and through dog-wolf crossbreeding rather than a separate domestication event. The high frequency of d1 suggests that the dog-wolf hybrid phenotype had a selective advantage.

  • 29.
    Lindh, Jenny M.
    et al.
    Dept of genetics microbiology and toxicology, Stockholm University.
    Terenius, Olle
    Dept of genetics microbiology and toxicology, Stockholm University.
    Faye, Ingrid
    Dept of genetics microbiology and toxicology, Stockholm University.
    16S rRNA gene-based identification of midgut bacteria from field-caught Anopheles gambiae sensu lato and A. funestus mosquitoes reveals new species related to known insect symbionts2005In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 71, no 11, p. 7217-7223Article in journal (Refereed)
    Abstract [en]

    Field-collected mosquitoes of the two main malaria vectors in Africa, Anopheles gambiae sensu lato and Anopheles funestus, were screened for their midgut bacterial contents. The midgut from each blood-fed mosquito was screened with two different detection pathways, one culture independent and one culture dependent. Bacterial species determination was achieved by sequence analysis of 16S rRNA genes. Altogether, 16 species from 14 genera were identified, 8 by each method. Interestingly, several of the bacteria identified are related to bacteria known to be symbionts in other insects. One isolate, Nocardia corynebacterioides, is a relative of the symbiont found in the vector for Chagas' disease that has been proven useful as a paratransgenic bacterium. Another isolate is a novel species within the gamma-proteobacteria that could not be phylogenetically placed within any of the known orders in the class but is close to a group of insect symbionts. Bacteria representing three intracellular genera were identified, among them the first identifications of Anaplasma species from mosquitoes and a new mosquito-Spiroplasma association. The isolates will be further investigated for their suitability for a paratransgenic Anopheles mosquito.

  • 30. Lindholm, Malene E.
    et al.
    Giacomello, Stefania
    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.
    Fischer, Helene
    Huss, Mikael
    Kjellqvist, Sanela
    Sundberg, Carl Johan
    The Impact of Endurance Training on Human Skeletal Muscle Memory, Global Isoform Expression and Novel Transcripts2016In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 12, no 9, article id e1006294Article in journal (Refereed)
    Abstract [en]

    Regularly performed endurance training has many beneficial effects on health and skeletal muscle function, and can be used to prevent and treat common diseases e.g. cardiovascular disease, type II diabetes and obesity. The molecular adaptation mechanisms regulating these effects are incompletely understood. To date, global transcriptome changes in skeletal muscles have been studied at the gene level only. Therefore, global isoform expression changes following exercise training in humans are unknown. Also, the effects of repeated interventions on transcriptional memory or training response have not been studied before. In this study, 23 individuals trained one leg for three months. Nine months later, 12 of the same subjects trained both legs in a second training period. Skeletal muscle biopsies were obtained from both legs before and after both training periods. RNA sequencing analysis of all 119 skeletal muscle biopsies showed that training altered the expression of 3,404 gene isoforms, mainly associated with oxidative ATP production. Fifty-four genes had isoforms that changed in opposite directions. Training altered expression of 34 novel transcripts, all with protein-coding potential. After nine months of detraining, no training-induced transcriptome differences were detected between the previously trained and untrained legs. Although there were several differences in the physiological and transcriptional responses to repeated training, no coherent evidence of an endurance training induced transcriptional skeletal muscle memory was found. This human lifestyle intervention induced differential expression of thousands of isoforms and several transcripts from unannotated regions of the genome. It is likely that the observed isoform expression changes reflect adaptational mechanisms and processes that provide the functional and health benefits of regular physical activity.

  • 31. Lourbakos, A.
    et al.
    Hiller, M.
    Kozaczynska, K.
    Baptiste, R. Jean
    Reza, M.
    Niks, E.
    Koeks, Z.
    de Klerk, D.
    Wolterbeek, R.
    Campion, G.
    Nadarajah, V. D.
    Szigyarto, Cristina Al-Khalili
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Calissano, M.
    Muntoni, F.
    Lochmueller, H.
    Verschuuren, J. J. G. M.
    Goemans, N.
    Tulinius, M.
    de Kimpe, S.
    Aartsma-Rus, A.
    't Hoen, P. A. C.
    Spitali, P.
    MMP-9 serum levels increase over time in Duchenne muscular dystrophy patients and decrease upon treatment with drisapersen2014In: Human Gene Therapy, ISSN 1043-0342, E-ISSN 1557-7422, Vol. 25, no 11, p. A27-A28Article in journal (Other academic)
  • 32. Maes, Alexandre
    et al.
    Céline, Gracia
    Innocenti, Nicolas
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST).
    Zhang, Kaiyang
    Aalto University, Finland .
    Aurell, Erik
    KTH, School of Computer Science and Communication (CSC), Computational Science and Technology (CST).
    Hajnsdorf, Eliane
    Landscape of RNA polyadenylation in E. coli2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
    Abstract [en]

    Polyadenylation is involved in degradation and quality control of bacterial RNAs. We used a combination of 5’-tagRACE and RNA-seq to analyse the total RNA content from wild-type strain and from mutant deficient for poly(A)polymerase. We determined that 157 mRNAs were affected as well as non-coding transcripts, up- and downregulated in the mutant when compared to the wild-type strain. Antisense RNAs were also detected and differentially affected by polyadenylation.

    Our results clearly reveal a correlation between the RNA folding energy and the requirement of polyadenylation to achieve the RNA decay. A new algorithm was developed to detect in both strains posttranscriptional modifications based on unmappable 3’-ends to analyse their position and composition. Therefore, any RNA 3'-end can be polyadenylated addressing them to the exoribonucleolytic machinery which is essential to degrade structured RNAs. Importantly, poly(A)polymerase was also upregulating the expression of genes related with the entire FliA regulon and numerous membrane transporters while downregulating the expression of the antigen 43 (flu), numerous sRNAs, antisense transcripts, REP sequences with the accumulation of numerous RNA fragments resulting from the processing of entire transcripts. Altogether we show here that polyadenylation has a broader spectrum of action than was suspected until now.

  • 33.
    Mardinoglu, Adil
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlen, Mathias
    Boren, Jan
    Broad Views of Non-alcoholic Fatty Liver Disease2018In: CELL SYSTEMS, ISSN 2405-4712, Vol. 6, no 1, p. 7-9Article in journal (Other academic)
    Abstract [en]

    Multi-omics multi-tissue data are used to interpret genome-wide association study results from mice to identify key driver genes of non-alcoholic fatty liver disease. Non-alcoholic fatty liver disease (NAFLD) is the accumulation of fat (steatosis) in the liver due to causes other than excessive alcohol consumption. The disease may progress to more severe forms of liver diseases, including non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. In this issue of Cell Systems, Krishnan et al. (2018) reveal mechanisms underlying NAFLD by generating multi-omics data using liver and adipose tissues obtained from the Hybrid Mouse Diversity Panel, consisting of 113 mouse strains with various degrees of NAFLD. The study identified key driver genes of NAFLD that can be used in the development of efficient treatment strategies and illustrates the potential utility of systematic analysis of multi-layer biological networks.

  • 34. Narooie-Nejad, Mehrnaz
    et al.
    Paylakhi, Seyed Hassan
    Shojaee, Seyedmehdi
    Fazlali, Zeinab
    Kanavi, Mozhgan Rezaei
    Nilforushan, Naveed
    Yazdani, Shahin
    Babrzadeh, Farbod
    National Institute of Genetic Engineering and Biotechnology, Tehran, Iran .
    Suri, Fatemeh
    Ronaghi, Mostafa
    Elahi, Elahe
    Paisan-Ruiz, Coro
    Loss of function mutations in the gene encoding latent transforming growth factor beta binding protein 2, LTBP2, cause primary congenital glaucoma2009In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 18, no 20, p. 3969-3977Article in journal (Refereed)
    Abstract [en]

    Glaucoma is a heterogeneous group of optic neuropathies that manifests by optic nerve head cupping or degeneration of the optic nerve, resulting in a specific pattern of visual field loss. Glaucoma leads to blindness if left untreated, and is considered the second leading cause of blindness worldwide. The subgroup primary congenital glaucoma (PCG) is characterized by an anatomical defect in the trabecular meshwork, and age at onset in the neonatal or infantile period. It is the most severe form of glaucoma. CYP1B1 was the first gene genetically linked to PCG, and CYP1B1 mutations are the cause of disease in 20-100% of patients in different populations. Here, we report that LTBP2 encoding latent transforming growth factor beta binding protein 2 is a PCG causing gene, confirming results recently reported. A disease-associated locus on chromosome 14 was identified by performing whole genome autozygosity mapping in Iranian PCG families using high density single nucleotide polymorphism chips, and two disease-segregating loss of function mutations in LTBP2, p.Ser472fsX3 and p.Tyr1793fsX55, were observed in two families while sequencing candidate genes in the locus. The p.Tyr1793fsX55 mutation affects an amino acid close to the C-terminal of the encoded protein. Subsequently, LTBP2 expression was shown in human eyes, including the trabecular meshwork and ciliary processes that are thought to be relevant to the etiology of PCG.

  • 35.
    Neiman, Mårten
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Tagging systems for sequencing large cohorts2010Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Advances in sequencing technologies constantly improves the throughput andaccuracy of sequencing instruments. Together with this development comes newdemands and opportunities to fully take advantage of the massive amounts of dataproduced within a sequence run. One way of doing this is by analyzing a large set ofsamples in parallel by pooling them together prior to sequencing and associating thereads to the corresponding samples using DNA sequence tags. Amplicon sequencingis a common application for this technique, enabling ultra deep sequencing andidentification of rare allelic variants. However, a common problem for ampliconsequencing projects is formation of unspecific PCR products and primer dimersoccupying large portions of the data sets.

    This thesis is based on two papers exploring these new kinds of possibilities andissues. In the first paper, a method for including thousands of samples in the samesequencing run without dramatically increasing the cost or sample handlingcomplexity is presented. The second paper presents how the amount of high qualitydata from an amplicon sequencing run can be maximized.

    The findings from the first paper shows that a two-tagging system, where the first tagis introduced by PCR and the second tag is introduced by ligation, can be used foreffectively sequence a cohort of 3500 samples using the 454 GS FLX Titaniumchemistry. The tagging procedure allows for simple and easy scalable samplehandling during sequence library preparation. The first PCR introduced tags, that arepresent in both ends of the fragments, enables detection of chimeric formation andhence, avoiding false typing in the data set.

    In the second paper, a FACS-machine is used to sort and enrich target DNA covered emPCR beads. This is facilitated by tagging quality beads using hybridization of afluorescently labeled target specific DNA probe prior to sorting. The system wasevaluated by sequencing two amplicon libraries, one FACS sorted and one standardenriched, on the 454 showing a three-fold increase of quality data obtained.

  • 36.
    Neiman, Mårten
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ahmadian, Afshin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Decoding a substantial set of samples in parallel by massive sequencing2011In: Plos One, ISSN 1932-6203, Vol. 6, no 3Article in journal (Refereed)
    Abstract [en]

    The dramatic increase of throughput seen in the eld of sequenceanalysis during the last years has opened up new possibilities of se-quencing a multitude of samples in parallel. Here we present a novelstrategy where the combination of two tags is used to link reads totheir origins in a pool of samples. The two tags are incorporated intwo steps leading to lowering of sample handling complexity by nearly100 times. The method described here enables accurate identi cationand typing of thousands of samples in parallel and is scalable. In thisstudy the system was designed to test 4992 samples using only 122 tags.

    To proof the concept of two tagging method the highly polymor-phic 2nd exon of DLA-DRB1 in dogs and wolves was sequenced usingthe 454 GS FLX Titanium Chemistry. By requiring a minimum se-quence depth of 20 reads per sample, 94% of the successfully ampli edsamples were genotyped. In addition, the method allowed digital de-tection of chimeric fragments. These results demonstrate that it ispossible to sequence thousands of samples in parallel without complexpooling patterns or primer combinations. Furthermore, the method isscalable and increasing the sample size by 960 samples requires only10 additional tags.

  • 37. Pang, Jun-Feng
    et al.
    Klütsch, Cornelya
    KTH, School of Biotechnology (BIO), Gene Technology.
    Zou, Xiao-Ju
    Zhang, Ai-bing
    KTH, School of Biotechnology (BIO), Gene Technology.
    Luo, Li-Yang
    Angleby, Helen
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ardalan, Arman
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ekström, Camilla
    KTH, School of Biotechnology (BIO), Gene Technology.
    Sköllermo, Anna
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Matsumura, Shuichi
    Leitner, Thomas
    Zhang, Ya-Ping
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    mtDNA Data Indicate a Single Origin for Dogs South of Yangtze River, Less Than 16,300 Years Ago, from Numerous Wolves2009In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 26, no 12, p. 2849-2864Article in journal (Refereed)
    Abstract [en]

    There is no generally accepted picture of where, when, and how the domestic dog originated. Previous studies of mitochondrial DNA (mtDNA) have failed to establish the time and precise place of origin because of lack of phylogenetic resolution in the so far studied control region (CR), and inadequate sampling. We therefore analyzed entire mitochondrial genomes for 169 dogs to obtain maximal phylogenetic resolution and the CR for 1,543 dogs across the Old World for a comprehensive picture of geographical diversity. Hereby, a detailed picture of the origins of the dog can for the first time be suggested. We obtained evidence that the dog has a single origin in time and space and an estimation of the time of origin, number of founders, and approximate region, which also gives potential clues about the human culture involved. The analyses showed that dogs universally share a common homogenous gene pool containing 10 major haplogroups. However, the full range of genetic diversity, all 10 haplogroups, was found only in southeastern Asia south of Yangtze River, and diversity decreased following a gradient across Eurasia, through seven haplogroups in Central China and five in North China and Southwest (SW)Asia, down to only four haplogroups in Europe. The mean sequence distance to ancestral haplotypes indicates an origin 5,400-16,300 years ago (ya) from at least 51 female wolf founders. These results indicate that the domestic dog originated in southern China less than 16,300 ya, from several hundred wolves. The place and time coincide approximately with the origin of rice agriculture, suggesting that the dogs may have originated among sedentary hunter-gatherers or early farmers, and the numerous founders indicate that wolf taming was an important culture trait.

  • 38. Peciulyte, Ausra
    et al.
    Anasontzis, George E.
    Karlström, Katarina
    Larsson, Per Tomas
    KTH, School of Chemical Science and Engineering (CHE), Centres, Wallenberg Wood Science Center. Innventia AB, Sweden.
    Olsson, Lisbeth
    Morphology and enzyme production of Trichoderma reesei Rut C-30 are affected by the physical and structural characteristics of cellulosic substrates2014In: Fungal Genetics and Biology, ISSN 1087-1845, E-ISSN 1096-0937, Vol. 72, p. 64-72Article in journal (Refereed)
    Abstract [en]

    The industrial production of cellulolytic enzymes is dominated by the filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina). In order to develop optimal enzymatic cocktail, it is of importance to understand the natural regulation of the enzyme profile as response to the growth substrate. The influence of the complexity of cellulose on enzyme production by the microorganisms is not understood. In the present study we attempted to understand how different physical and structural properties of cellulose-rich substrates affected the levels and profiles of extracellular enzymes produced by T. reesei. Enzyme production by T. reesei Rut C-30 was studied in submerged cultures on five different cellulose-rich substrates, namely, commercial cellulose Avicel (R) and industrial-like cellulosic pulp substrates which consist mainly of cellulose, but also contain residual hemicellulose and lignin. In order to evaluate the hydrolysis of the substrates by the fungal enzymes, the spatial polymer distributions were characterised by cross-polarisation magic angle spinning carbon-13 nuclear magnetic resonance (CP/MAS C-13-NMR) in combination with spectral fitting. Proteins in culture supernatants at early and late stages of enzyme production were labeled by Tandem Mass Tags (TMT) and protein profiles were analysed by liquid chromatography-tandem mass spectrometry. The data have been deposited to the ProteomeXchange with identifier PXD001304. In total 124 proteins were identified and quantified in the culture supernatants, including cellulases, hemicellulases, other glycoside hydrolases, lignin-degrading enzymes, auxiliary activity 9 (AA9) family (formerly GH61), supporting activities of proteins and enzymes acting on cellulose, proteases, intracellular proteins and several hypothetical proteins. Surprisingly, substantial differences in the enzyme profiles were found even though there were minor differences in the chemical composition between the cellulose-rich substrates.

  • 39. Quince, Christopher
    et al.
    Delmont, Tom O.
    Raguideau, Sebastien
    Alneberg, Johannes
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Darling, Aaron E.
    Collins, Gavin
    Eren, A. Murat
    DESMAN: a new tool for de novo extraction of strains from metagenomes2017In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 18, article id 181Article in journal (Refereed)
    Abstract [en]

    We introduce DESMAN for De novo Extraction of Strains from Metagenomes. Large multi-sample metagenomes are being generated but strain variation results in fragmentary co-assemblies. Current algorithms can bin contigs into metagenome-assembled genomes but are unable to resolve strain-level variation. DESMAN identifies variants in core genes and uses co-occurrence across samples to link variants into haplotypes and abundance profiles. These are then searched for against non-core genes to determine the accessory genome of each strain. We validated DESMAN on a complex 50-species 210-genome 96-sample synthetic mock data set and then applied it to the Tara Oceans microbiome.

  • 40. Sakthivel, Priya
    et al.
    Wang, Xiongbiao
    Gharizadeh, Baback
    Giscombe, Ricardo
    Pirskanen, Ritva
    Nyrén, Pål
    KTH, School of Biotechnology (BIO), Biochemistry (closed 20130101).
    Lefvert, Ann Kari
    Single-nucleotide polymorphisms in the B7H3 gene are not associated with human autoimmune myasthenia gravis2006In: Journal of Genetics, ISSN 0022-1333, E-ISSN 0973-7731, Vol. 85, no 3, p. 217-220Article in journal (Refereed)
  • 41. Shirley, B. C.
    et al.
    Mucaki, E. J.
    Whitehead, T.
    Costea, Paul Igor
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Akan, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rogan, P. K.
    Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences2013In: Genomics, Proteomics and Bioinformatics, ISSN 1672-0229, Vol. 11, no 2, p. 77-85Article in journal (Refereed)
    Abstract [en]

    Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.

  • 42.
    Sigurgeirsson, Benjamin
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).
    Åmark, Hanna
    Karolinska Inst, Dept Clin Sci & Educ, Unit Obstet & Gynecol, Sjukhusbacken 10, S-11883 Stockholm, Sweden.
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ujvari, Dorina
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden.
    Westgren, Magnus
    Karolinska Inst, Dept Clin Sci Intervent & Technol, Stockholm, Sweden.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gidlöf, Sebastian
    Karolinska Inst, Dept Womens & Childrens Hlth, Stockholm, Sweden;Karolinska Inst, Dept Clin Sci Intervent & Technol, Stockholm, Sweden;Karolinska Univ Hosp Huddinge, Dept Obstet & Gynecol, Stockholm, Sweden.
    Comprehensive RNA sequencing of healthy human endometrium at two time points of the menstrual cycle2017In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 96, no 1, p. 24-33Article in journal (Refereed)
    Abstract [en]

    Endometrial receptivity is crucial for implantation and establishment of a normal pregnancy. The shift from proliferative to receptive endometrium is still far from being understood. In this paper, we comprehensively present the transcriptome of the human endometrium by comparing endometrial biopsies from proliferative phase with consecutive biopsies 7-9 days after ovulation. The results show a clear difference in expression between the two time points using both total and small RNA sequencing. A total of 3,297 messenger RNAs (mRNAs), 516 long noncoding RNAs (lncRNAs), and 102 small noncoding RNAs were identified as statistically differentially expressed between the two time points. We show a thorough description of the change in mRNA between the two time points and display lncRNAs, small nucleolar RNAs, and small nuclear RNAs not previously reported in the healthy human endometrium. In conclusion, this paper reports in detail the shift in RNA expression from the proliferative to receptive endometrium. Summary Sentence Messenger RNA, sncRNA, and lncRNA show a clear difference in expression between proliferative phase and 7-9 days after ovulation, thoroughly described together with lncRNA, snoRNA, and snRNA not previously reported in healthy human endometrium.

  • 43. Sillén, Anna
    et al.
    Brohede, Jesper
    Forsell, Charlotte
    Lilius, Lena
    Andrade, Jorge
    KTH, School of Biotechnology (BIO).
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Proteomics.
    Kimura, Toru
    Winblad, Bengt
    Graff, Caroline
    Linkage Analysis of Autopsy-Confirmed Familial Alzheimer Disease Supports an Alzheimer Disease Locus in 8q242011In: Dementia and Geriatric Cognitive Disorders, ISSN 1420-8008, E-ISSN 1421-9824, Vol. 31, no 2, p. 109-118Article in journal (Refereed)
    Abstract [en]

    Background/Aims: We have previously reported the results of an extended genome-wide scan of Swedish Alzheimer disease (AD)-affected families; in this paper, we analyzed a subset of these families with autopsy-confirmed AD. Methods: We report the fine-mapping, using both microsatellite markers and single-nucleotide polymorphisms (SNPs), in the observed maximum logarithm of the odds (LOD)-2 unit (LODmax-2) region under the identified linkage peak, linkage analysis of the fine-mapping data with additionally analyzed pedigrees, and association analysis of SNPs selected from candidate genes in the linked interval. The subset was made on the criterion of at least one autopsy-confirmed AD case per family, resulting in 24 families. Results: Linkage analysis of a family subset having at least one autopsy-confirmed AD case showed a significant nonparametric single-point LOD score of 4.4 in 8q24. Fine-mapping under the linkage peak with 10 microsatellite markers yielded an increase in the multipoint (mpt) LOD score from 2.1 to 3.0. SNP genotyping was performed on 21 selected candidate transcripts of the LODmax-2 region. Both family-based association and linkage analysis were performed on extended material from 30 families, resulting in a suggestive linkage at peak marker rs6577853 (mpt LOD score = 2.4). Conclusion: The 8q24 region has been implicated to be involved in AD etiology.

  • 44.
    Stahl, Patrik L.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Salmen, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundmark, Anna
    KTH, School of Biotechnology (BIO), Gene Technology.
    Navarro, Jose Fernandez
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Magnusson, Jens
    Giacomello, Stefania
    KTH, School of Biotechnology (BIO), Gene Technology.
    Asp, Michaela
    Westholm, Jakub O.
    Huss, Mikael
    Mollbrink, Annelie
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Linnarsson, Sten
    Codeluppi, Simone
    Borg, Ake
    Ponten, Fredrik
    Costea, Paul Igor
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlen, Pelin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mulder, Jan
    Bergmann, Olaf
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisen, Jonas
    Visualization and analysis of gene expression in tissue sections by spatial transcriptomics2016In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 353, no 6294, p. 78-82Article in journal (Refereed)
    Abstract [en]

    Analysis of the pattern of proteins or messenger RNAs (mRNAs) in histological tissue sections is a cornerstone in biomedical research and diagnostics. This typically involves the visualization of a few proteins or expressed genes at a time. We have devised a strategy, which we call "spatial transcriptomics," that allows visualization and quantitative analysis of the transcriptome with spatial resolution in individual tissue sections. By positioning histological sections on arrayed reverse transcription primers with unique positional barcodes, we demonstrate high-quality RNA-sequencing data with maintained two-dimensional positional information from the mouse brain and human breast cancer. Spatial transcriptomics provides quantitative gene expression data and visualization of the distribution of mRNAs within tissue sections and enables novel types of bioinformatics analyses, valuable in research and diagnostics.

  • 45.
    Ståhl, Patrik L.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Methods for Analyzing Genomes2010Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human genome reference sequence has given us a two‐dimensional blueprint of our inherited code of life, but we need to employ modern‐day technology to expand our knowledge into a third dimension. Inter‐individual and intra‐individual variation has been shown to be larger than anticipated, and the mode of genetic regulation more complex. Therefore, the methods that were once used to explain our fundamental constitution are now used to decipher our differences. Over the past four years, throughput from DNA‐sequencing platforms has increased a thousand‐fold, bearing evidence of a rapid development in the field of methods used to study DNA and the genomes it constitutes. The work presented in this thesis has been carried out as an integrated part of this technological evolution, contributing to it, and applying the resulting solutions to answer difficult biological questions.

    Papers I and II describe a novel approach for microarray readout based on immobilization of magnetic particles, applicable to diagnostics. As benchmarked on canine mitochondrial DNA, and human genomic DNA from individuals with cystic fibrosis, it allows for visual interpretation of genotyping results without the use of machines or expensive equipment. Paper III outlines an automated and cost‐efficient method for enrichment and titration of clonally amplified DNA‐libraries on beads. The method uses fluorescent labeling and a flow‐cytometer to separate DNA‐beads from empty ones. At the same time the fraction of either bead type is recorded, and a titration curve can be generated. In paper IV we combined the highly discriminating multiplex genotyping of trinucleotide threading with the digital readout made possible by massively parallel sequencing. From this we were able to characterize the allelic distribution of 88 obesity related SNPs in a population of 462 individuals enrolled at a childhood obesity center. Paper V employs the throughput of present day DNA sequencingas it investigates deep into sun‐exposed skin to find clues on the effects of sunlight during the course of a summer holiday. The tumor suppressor p53 gene was targeted, only to find that despite its well‐documented involvement in the disease progression of cancers, an estimated 35,000 novel sun‐induced persistent p53 mutations are added and phenotypically tolerated in the skin of every individual every year. The last paper, VI, describes a novel approach for finding breast cancer biomarkers. In this translational study we used differential protein expression profiles and sequence capture to select and enrich for 52 candidate genes in DNA extracted from ten tumors. Two of the genes turned out to harbor protein‐altering mutations in multiple individuals.

  • 46. Svedberg, Anna
    et al.
    Green, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Linkoping Univ, Dept Med & Hlth Sci, Div Drug Res, Clin Pharmacol, SE-58185 Linkoping, Sweden.
    Vikström, Anders
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vikingsson, Svante
    A validated liquid chromatography tandem mass spectrometry method for quantification of erlotinib, OSI-420 and didesmethyl erlotinib and semi-quantification of erlotinib metabolites in human plasma2015In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 107, p. 186-195Article in journal (Refereed)
    Abstract [en]

    A liquid chromatography tandem mass spectrometry method was developed and validated for quantification of erlotinib and its metabolites in human plasma. The method is suitable for therapeutic drug monitoring and pharmacokinetic studies. The substances were extracted using protein precipitation, separated on a BEH XBridge C18 column (100 x 2.1 mm, 1.7 mu m) by gradient elution at 0.7 mL/min of acetonitrile and 5 mM ammonium acetate. The concentration was determined using a Waters Xevo triple quadrupole mass spectrometer in a multi reaction monitoring mode. The total run time was 7 min. Deuterated erlotinib and OSI-597 were used as internal standard for erlotinib and its metabolites, respectively. Erlotinib, OSI-420 and didesmethyl erlotinib were quantified in the concentration range 25-5000 ng/mL, 0.5-500 ng/mL and 0.15-10 ng/mL, respectively. Precision and accuracy was <14% except for OSI-420 at LLOQ (17%). Extraction recovery was above 89%, 99% and 89% for erlotinib, OSI-420 and didesmethyl erlotinib, respectively. The human liver microsomes generated 14 metabolites, three of them not previously reported. Twelve metabolites were measured semi-quantitatively and validated with respect to selectivity, precision and stability.

  • 47.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Three-dimensional whole transcriptome analysis of tissue for classification of breast cancer2017Manuscript (preprint) (Other academic)
  • 48.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Three-dimensional whole transcriptome analysis of tissue for classification of breast cancer. Submitted manuscript.2017Manuscript (preprint) (Other academic)
  • 49.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Transcriptome-wide analysis in cells and tissues2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    High-throughput sequencing has greatly influenced the amount of data produced and biological questions asked and answered. Sequencing approaches have also enabled rapid development of related technological fields such as single-cell and spatially resolved expression profiling. The introductory parts of this thesis give an overview of the basic molecular and technological apparatus needed to analyse the transcriptome in cells and tissues. This is succeeded by a summary of present investigations that report recent advancements in RNA profiling.

    RNA integrity needs to be preserved for accurate gene expression analysis. A method providing a low-cost alternative for RNA preservation was reported. Namely, a low concentration of buffered formaldehyde was used for fixation of human cell lines and peripheral blood cells (Paper I). The results from bulk RNA sequencing confirmed gene expression was not negatively impacted with the preservation procedure (r2>0.88) and that long-term storage of such samples was possible (r2=0.95). However, it is important to note that a small population of cells overexpressing a limited amount of genes can skew bulk gene expression analyses making them sufficient only in carefully designed studies. Therefore, gene expression should be investigated at the single cell resolution when possible. A method for high-throughput single cell expression profiling termed microarrayed single-cell sequencing was developed (Paper II). The method incorporated fluorescence-activated cell sorting, sample deposition and profiling of thousands of barcoded single cells in one reaction. After sample attachment to a barcoded array, a high-resolution image was taken which linked the position of each array barcode sequence to each individual deposited cell. The cDNA synthesis efficiency was estimated at 17.3% while detecting 27,427 transcripts per cell on average. Additionally, spatially resolved analysis is important in cell differentiation, organ development and pathological changes. Current methods are limited in terms of throughput, cost and time. For that reason, the spatial transcriptomics method was developed (Paper III). Here, the barcoded microarray was used to obtain spatially resolved expression profiles from tissue sections using the same imaging principle. The mouse olfactory bulb was profiled on a whole-transcriptome scale and the results showed that the expression correlated well (r2=0.94-0.97) as compared to bulk RNA sequencing. The method was 6.9% efficient, reported signal diffusion at ~2 μm and accurately deconvoluted layer-specific transcripts in an unbiased manner. Lastly, the spatial transcriptomics concept was applied to profile human breast tumours in three dimensions (Paper IV). Unbiased clustering revealed previously un-annotated regions and classified them as parts of the immune system, providing a detailed view into complex interactions and crosstalk in the whole tissue volume. Spatial tumour classification divulged that certain parts of the tumour clearly classified as other subtypes as compared to bulk analysis providing useful data for current practice diagnostics.

    The last part of the thesis discusses a look towards the future, how the presented methods could be used, improved upon or combined in translational research.

  • 50. Wang, Chunlin
    et al.
    Sanders, Catherine M.
    Yang, Qunying
    Schroeder, Harry W., Jr.
    Wang, Elijah
    Babrzadeh, Farbod
    Stanford Genome Technology Center, United States .
    Gharizadeh, Baback
    Myers, Richard M.
    Hudson, James R., Jr.
    Davis, Ronald W.
    Han, Jian
    High throughput sequencing reveals a complex pattern of dynamic interrelationships among human T cell subsets2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 4, p. 1518-1523Article in journal (Refereed)
    Abstract [en]

    Developing T cells face a series of cell fate choices in the thymus and in the periphery. The role of the individual T cell receptor (TCR) in determining decisions of cell fate remains unresolved. The stochastic/selection model postulates that the initial fate of the cell is independent of TCR specificity, with survival dependent on additional TCR/coreceptor "rescue" signals. The "instructive" model holds that cell fate is initiated by the interaction of the TCR with a cognate peptide-MHC complex. T cells are then segregated on the basis of TCR specificity with the aid of critical coreceptors and signal modulators [Chan S, Correia-Neves M, Benoist C, Mathis (1998) Immunol Rev 165: 195-207]. The former would predict a random representation of individual TCR across divergent T cell lineages whereas the latter would predict minimal overlap between divergent T cell subsets. To address this issue, we have used high-throughput sequencing to evaluate the TCR distribution among key T cell developmental and effector subsets from a single donor. We found numerous examples of individual subsets sharing identical TCR sequence, supporting a model of a stochastic process of cell fate determination coupled with dynamic patterns of clonal expansion of T cells bearing the same TCR sequence among both CD4(+) and CD8+ populations.

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