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  • 251. Naett, Daniel
    et al.
    Lindqvist, Niclas
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Torjesen, Peter A.
    Jensen, Per
    Inheritance of Acquired Behaviour Adaptations and Brain Gene Expression in Chickens2009Article in journal (Refereed)
    Abstract [en]

    Background: Environmental challenges may affect both the exposed individuals and their offspring. We investigated possible adaptive aspects of such cross-generation transmissions, and hypothesized that chronic unpredictable food access would cause chickens to show a more conservative feeding strategy and to be more dominant, and that these adaptations would be transmitted to the offspring. Methodology/Principal Findings: Parents were raised in an unpredictable (UL) or in predictable diurnal light rhythm (PL, 12:12 h light: dark). In a foraging test, UL birds pecked more at freely available, rather than at hidden and more attractive food, compared to birds from the PL group. Female offspring of UL birds, raised in predictable light conditions without parental contact, showed a similar foraging behavior, differing from offspring of PL birds. Furthermore, adult offspring of UL birds performed more food pecks in a dominance test, showed a higher preference for high energy food, survived better, and were heavier than offspring of PL parents. Using cDNA microarrays, we found that the differential brain gene expression caused by the challenge was mirrored in the offspring. In particular, several immunoglobulin genes seemed to be affected similarly in both UL parents and their offspring. Estradiol levels were significantly higher in egg yolk from UL birds, suggesting one possible mechanism for these effects. Conclusions/Significance: Our findings suggest that unpredictable food access caused seemingly adaptive responses in feeding behavior, which may have been transmitted to the offspring by means of epigenetic mechanisms, including regulation of immune genes. This may have prepared the offspring for coping with an unpredictable environment.

  • 252.
    Natanaelsson, Christian
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Oskarsson, Mattias C. R.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Angleby, Helen
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Kirkness, Ewen
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Dog Y chromosomal DNA sequence: identification, sequencing and SNP discovery2006In: BMC Genetics, ISSN 1471-2156, E-ISSN 1471-2156, Vol. 7Article in journal (Refereed)
    Abstract [en]

    Background: Population genetic studies of dogs have so far mainly been based on analysis of mitochondrial DNA, describing only the history of female dogs. To get a picture of the male history, as well as a second independent marker, there is a need for studies of biallelic Y-chromosome polymorphisms. However, there are no biallelic polymorphisms reported, and only 3200 bp of non-repetitive dog Y-chromosome sequence deposited in GenBank, necessitating the identification of dog Y chromosome sequence and the search for polymorphisms therein. The genome has been only partially sequenced for one male dog, disallowing mapping of the sequence into specific chromosomes. However, by comparing the male genome sequence to the complete female dog genome sequence, candidate Y-chromosome sequence may be identified by exclusion. Results: The male dog genome sequence was analysed by Blast search against the human genome to identify sequences with a best match to the human Y chromosome and to the female dog genome to identify those absent in the female genome. Candidate sequences were then tested for male specificity by PCR of five male and five female dogs. 32 sequences from the male genome, with a total length of 24 kbp, were identified as male specific, based on a match to the human Y chromosome, absence in the female dog genome and male specific PCR results. 14437 bp were then sequenced for 10 male dogs originating from Europe, Southwest Asia, Siberia, East Asia, Africa and America. Nine haplotypes were found, which were defined by 14 substitutions. The genetic distance between the haplotypes indicates that they originate from at least five wolf haplotypes. There was no obvious trend in the geographic distribution of the haplotypes. Conclusion: We have identified 24159 bp of dog Y-chromosome sequence to be used for population genetic studies. We sequenced 14437 bp in a worldwide collection of dogs, identifying 14 SNPs for future SNP analyses, and giving a first description of the dog Y-chromosome phylogeny.

  • 253.
    Navarro, Jose Fernandez
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sjöstrand, Joel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Salmén, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ståhl, Patrik L.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institutet, Sverige.
    ST Pipeline: an automated pipeline for spatial mapping of unique transcripts2017In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 33, no 16, p. 2591-2593Article in journal (Refereed)
    Abstract [en]

    Motivation: In recent years we have witnessed an increase in novel RNA-seq based techniques for transcriptomics analysis. Spatial transcriptomics is a novel RNA-seq based technique that allows spatial mapping of transcripts in tissue sections. The spatial resolution adds an extra level of complexity, which requires the development of new tools and algorithms for efficient and accurate data processing. Results: Here we present a pipeline to automatically and efficiently process RNA-seq data obtained from spatial transcriptomics experiments to generate datasets for downstream analysis.

  • 254.
    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.

  • 255.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mathematical modeling of yeast: a driver for innovation in biotechnology and human medicine2015In: Yeast, ISSN 0749-503X, E-ISSN 1097-0061, Vol. 32, p. S50-S51Article in journal (Other academic)
    Abstract [en]

    Saccharomyces cerevisiae is the most studied organism and is for this reason used widely as a model organismfor studying molecular mechanisms of relevance for human disease. Thus, several Nobel Prizes have been givento yeast researchers. Among many other discoveries studies of yeast resulted in identification of cyclins andcyclin dependent kinases that play a central role in the cell cycle of eukaryal cells and mapping of the proteinsecretory pathway in eukaryal cells. This yeast is also used for production of fuels, chemicals, pharmaceuticalsand materials, and the annual revenue derived from processes based on S. cerevisiae fermentations by farexceeds 200 billion EURO. Furthermore. through metabolic engineering of this yeast a number of novel newindustrial processes are under development resulting in an even more important role of this cell factory in thefuture. In order to advance our fundamental understanding of this important organism, but in human healthresearch and industrial biotechnology, it is important to advance our ability to integrate novel experimental datain quantitative framework. Mathematical models represent an excellent scaffold for this as they allowreconciliation of data and at the same time enable generation of novel hypothesis concerning specific molecularprocesses. Furthermore, in the field of industrial biotechnology mathematical models may be used for advancingmetabolic engineering, which will result in a reduction in development costs and hereby advance towards biosustainable production of fuels and chemicals

  • 256.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology.
    Yeast as a platform cell factory in future biorefineries2013In: 4th International Conference on Biomolecular Engineering, ICBE 2013, 2013, p. 56-79Conference paper (Refereed)
  • 257.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers, Dept Biol & Biol Engn, SE-41296 Gothenburg, Sweden.;Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2970 Horsholm, Denmark.;Royal Inst Technol, Sci Life Lab, SE-17121 Stockholm, Sweden..
    Yeast cell factories on the horizon2015In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 349, no 6252, p. 1050-1051Article in journal (Other academic)
  • 258.
    Nielsen, Jens
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fussenegger, Martin
    Keasling, Jay
    Lee, Sang Yup
    Liao, James C.
    Prather, Kristala
    Palsson, Bernhard
    Engineering synergy in biotechnology2014In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 10, no 5, p. 319-322Article in journal (Refereed)
  • 259.
    Nielsen, Jens
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Department of Biology and Biological Engineering, Chalmers University of Technology, Sweden.
    Keasling, Jay D.
    Engineering Cellular Metabolism2016In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 164, no 6, p. 1185-1197Article, review/survey (Refereed)
    Abstract [en]

    Metabolic engineering is the science of rewiring the metabolism of cells to enhance production of native metabolites or to endow cells with the ability to produce new products. The potential applications of such efforts are wide ranging, including the generation of fuels, chemicals, foods, feeds, and pharmaceuticals. However, making cells into efficient factories is challenging because cells have evolved robust metabolic networks with hard-wired, tightly regulated lines of communication between molecular pathways that resist efforts to divert resources. Here, we will review the current status and challenges of metabolic engineering and will discuss how new technologies can enable metabolic engineering to be scaled up to the industrial level, either by cutting off the lines of control for endogenous metabolism or by infiltrating the system with disruptive, heterologous pathways that overcome cellular regulation.

  • 260. Nilsson, D.
    et al.
    Pettersson, M.
    Gustavsson, P.
    Förster, A.
    Hofmeister, W.
    Wincent, J.
    Zachariadis, V.
    Anderlid, B. -M
    Nordgren, A.
    Mäkitie, O.
    Wirta, Valtteri
    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.
    Vezzi, F.
    Lupski, J. R.
    Nordenskjöld, M.
    Syk Lundberg, E.
    Carvalho, C. M. B.
    Lindstrand, A.
    Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation2017In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 38, no 2, p. 180-192Article in journal (Refereed)
    Abstract [en]

    Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low-coverage mate pair whole-genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX-male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error-prone replication-based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.

  • 261. Niskanen, A. K.
    et al.
    Hagström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lohi, H.
    Ruokonen, M.
    Esparza-Salas, R.
    Aspi, J.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    MHC variability supports dog domestication from a large number of wolves: high diversity in Asia2013In: Heredity, ISSN 0018-067X, E-ISSN 1365-2540, Vol. 110, no 1, p. 80-85Article in journal (Refereed)
    Abstract [en]

    The process of dog domestication is still somewhat unresolved. Earlier studies indicate that domestic dogs from all over the world have a common origin in Asia. So far, major histocompatibility complex (MHC) diversity has not been studied in detail in Asian dogs, although high levels of genetic diversity are expected at the domestication locality. We sequenced the second exon of the canine MHC gene DLA-DRB1 from 128 Asian dogs and compared our data with a previously published large data set of MHC alleles, mostly from European dogs. Our results show that Asian dogs have a higher MHC diversity than European dogs. We also estimated that there is only a small probability that new alleles have arisen by mutation since domestication. Based on the assumption that all of the currently known 102 DLA-DRB1 alleles come from the founding wolf population, we simulated the number of founding wolf individuals. Our simulations indicate an effective population size of at least 500 founding wolves, suggesting that the founding wolf population was large or that backcrossing has taken place.

  • 262. Nizamlioglu, M.
    et al.
    Bulut, Z.
    Erdogan, M.
    Tepeli, C.
    Yilmaz, A.
    Kurar, E.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genetic characterization of Akbas shepherd dogs in Turkey, Uzbekistan and Iran using STR markers2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, p. 349-349Article in journal (Other academic)
  • 263.
    Nystedt, Björn
    et al.
    Stockholm University.
    Vezzi, Francesco
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Alekseenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sahlin, Kristoffer
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hällman, Jimmie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rilakovic, Nemanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    et, al,
    The Norway spruce genome sequence and conifer genome evolution2013In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 497, no 7451, p. 579-584Article in journal (Refereed)
    Abstract [en]

    Conifers have dominated forests for more than 200 million years and are of huge ecological and economic importance. Here we present the draft assembly of the 20-gigabase genome of Norway spruce (Picea abies), the first available for any gymnosperm. The number of well-supported genes (28,354) is similar to the >100 times smaller genome of Arabidopsis thaliana, and there is no evidence of a recent whole-genome duplication in the gymnosperm lineage. Instead, the large genome size seems to result from the slow and steady accumulation of a diverse set of long-terminal repeat transposable elements, possibly owing to the lack of an efficient elimination mechanism. Comparative sequencing of Pinus sylvestris, Abies sibirica, Juniperus communis, Taxus baccata and Gnetum gnemon reveals that the transposable element diversity is shared among extant conifers. Expression of 24-nucleotide small RNAs, previously implicated in transposable element silencing, is tissue-specific and much lower than in other plants. We further identify numerous long (>10,000 base pairs) introns, gene-like fragments, uncharacterized long non-coding RNAs and short RNAs. This opens up new genomic avenues for conifer forestry and breeding.

  • 264. Odeberg, J. M.
    et al.
    Andrade, J.
    Holmberg, K.
    Hoglund, P.
    Malmqvist, U.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    UGT1A polymorphisms in a Swedish cohort and a human diversity panel, and the relation to bilirubin plasma levels in males and females2006In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 62, no 10, p. 829-837Article in journal (Refereed)
    Abstract [en]

    Objectives: The objective of this study was to investigate the prevalence of different polymorphisms and haplotypes associated with individual variations in pharmacokinetics and drug toxicity in the uridine-diphosphate glucuronosyl transferase (UGT) 1A gene in a Swedish cohort (248 healthy volunteers) and in 14 different ethnic groups. We also estimated UGT1A genotype-dependent glucuronidation efficiency using the endogenous substrate bilirubin as an indicator. Methods: Pyrosequencing-based genotyping assays were used to determine the different polymorphisms and haplotypes. Results: Haplotype analysis of the UGT1A1 (*1*28), UGT1A6 (*1*2), and UGT1A7(*1*2*3*4) allelic variants showed that three major haplotypes constituted 84% of the allelic variants in the cohort. We identified 15 haplotypes altogether from all groups, including previously undescribed haplotypes.Testing for the association of genotype and total bilirubin levels (nonfasting) in plasma disclosed that homozygous carriers of the TA allele, irrespective of haplotype combinations, had increased levels of bilirubin compared with noncarriers, but a gender-associated difference was observed. Conclusions: In a Swedish cohort, several genetic variants in the UGT1A gene are common, but prevalence in a population may differ because of ethnicity. A phenotype based on bilirubin levels has limitations in serving as an indicator of pharmacogenetic differences in glucuronidation due to the influence of gender. Because of possible substrate overlap regarding different UGT1A isoforms, determination of haplotypes of potential cis-acting polymorphisms in the UGT1A gene should be considered in pharmacogenetic association studies regarding drugs that undergo glucuronidation.

  • 265.
    Odeberg, Jacob
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Freitag, M.
    Odeberg, H.
    Rastam, L.
    Lindblad, U.
    Severity of acute coronary syndrome is predicted by interactions between fibrinogen concentrations and polymorphisms in the GPIIIa and FXIII genes2006In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836, Vol. 4, no 4, p. 909-912Article in journal (Refereed)
  • 266.
    Odeberg, Jacob
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Larsson, Charlotte A.
    Rastam, Lennart
    Lindblad, Ulf
    The Asp(298) allele of endothelial nitric oxide synthase is a risk factor for myocardial infarction among patients with type 2 diabetes mellitus2008In: BMC Cardiovascular Disorders, ISSN 1471-2261, E-ISSN 1471-2261, Vol. 8Article in journal (Refereed)
    Abstract [en]

    Background: Endothelial dysfunction plays a central role in atherosclerotic progression and cardiovascular complications of type 2 diabetes mellitus (T2DM). Given the role of nitric oxide in the vascular system, we aimed to test hypotheses of synergy between the common endothelial nitric oxide synthase (eNOS) Asp(298) allele and T2DM in predisposing to acute myocardial infarction (AMI). Methods: In a population-based patient survey with 403 persons with T2DM and 799 healthy subjects from the population without diabetes or hypertension, we analysed the relation between T2DM, sex and the eNOS Asp(298) allele versus the risk for AMI. Results: In an overall analysis, T2DM was a significant independent risk factor for AMI. In patients with T2DM, homozygosity for the eNOS Asp(298) allele was a significant risk factor (HR 3.12 [1.49-6.56], p = 0.003), but not in subjects without diabetes or hypertension. Compared to wild-type non-diabetic subjects, all patients with T2DM had a significantly increased risk of AMI regardless of genotype. This risk was however markedly higher in patients with T2DM homozygous for the Asp(298) allele (HR 7.20 [3.01-17.20], p < 0.001), independent of sex, BMI, systolic blood pressure, serum triglycerides, HDL -cholesterol, current smoking, and leisure time physical activity. The pattern seemed stronger in women than in men. Conclusion: We show here a strong independent association between eNOS genotype and AMI in patients with T2DM. This suggests a synergistic effect of the eNOS Asp(298) allele and diabetes, and confirms the role of eNOS as an important pathological bottleneck for cardiovascular disease in patients with T2DM.

  • 267.
    Oskarsson, Mattias
    KTH, School of Biotechnology (BIO), Gene Technology.
    Analysis of the origin and spread of the domestic dog using Y-chromosome DNA and mtDNA sequence data2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The domestic dog was probably the first domesticated animal, and the only one to spread to all continents in ancient times. The dog is one of the most phenotypically diverse animals, a result of human selection throughout dog history. Studies of the genetic origins and early spread of domestic dogs is important to gather information about biological and cultural mechanisms behind domestication but also to investigate early human history. The step from a hunter and gatherer lifestyle to farming is one of the most important steps in human history. In this thesis I will present work aimed at understanding both domestic dog origins and dispersal. In order to be able to investigate dog origins based on a second haploid chromosome we identified 14,437 bp of Y-chromosomal DNA sequence. Based on this we show that dogs in Asia south of Yangtze River (ASY) has the highest genetic diversity and was founded from a large number of wolf founders confirming earlier mtDNA results. Early dog dispersal is tightly coupled to human history with the dog brought along as a cultural item. We have for the first time investigated the dog dispersal into Polynesia and Australia and our data can be used as evidence for a more complex settlement of Polynesia than earlier indicated from archaeological and linguistic studies. Analysis of Y-chromosome SNPs in Australian dingoes confirms earlier mtDNA genetic studies that the dingo is part of the domestic dog phylogeny and was founded from a small population of domestic dogs. We have also for the first time investigated the dog population on Madagascar and our data strongly indicates a mainland African origin for the Madagascan dogs. Finally, we have investigated the American dog population sampled from throughout the continent and also for the first time included putative indigenous breed dogs such as Chihuahua and Pero Sín Pelo del Peru, and the free-ranging Carolina dogs from southern USA. Our data clearly indicates a primarily Old World origin for the indigenous breed dogs and also for the free-ranging Carolina dogs in USA. We can also for the first time present evidence for continuity between the ancient and extant dog population with e.g. exclusive sharing of a haplotype between a modern sample of Chihuahua and an ancient Mexican sample.

  • 268.
    Oskarsson, Mattias
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ardalan, Arman
    KTH, School of Biotechnology (BIO), Gene Technology.
    Rabakonandriania, Elisabeth
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    African origin for Madagascan dogs revealed by mtDNA analysisManuscript (preprint) (Other academic)
    Abstract [en]

    Dogs were the only domestic animals accompanying humans to every continent in ancient time, and was along with pig and chicken part of the Austronesian Neolithic culture. Madagascar was one of the last major land masses to be occupied by humans, 1,500-2,000 years ago. It was initially colonized by Austronesian speaking Indonesians but subsequent migration from Africa has resulted in approximately equal genetic contribution from Indonesia and Africa, and the material culture has mainly African influences. To track the initial worldwide dispersal of dogs and illuminate this part of Madagascan cultural origins we here investigate the ancestry of Malagasy dogs. We analysed mtDNA control region sequences in dogs from Madagascar (n=145) and compared with dogs from potential ancestral populations in Island Southeast Asia (n=219) and sub-Saharan Africa (n=493). We found that 90% of the Madagascan dogs carried a haplotype present also in sub-Saharan Africa, and the remaining lineages could all be attributed to a likely origin in Africa. In contrast, only 26% of Malagasy dogs shared a haplotype with Indonesian dogs, all of which universally occurring haplotypes, and three haplotypes typical for Austronesian dogs, carried by >50% of Indonesian and Polynesian dogs, were carried by only 1% of the Madagscan dogs. Thus, in contrast to the human population, the Madagscan dogs seem to trace its origin entirely from Africa. This indicates that dogs were not brought with the initial Austronesian speaking colonizers on their trans-ocean voyage but introduced at a later stage with the migration and cultural influence from Africa.

  • 269.
    Oskarsson, Mattias C. R.
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Klütsch, Cornelya F. C.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Boonyaprakob, Ukadej
    Wilton, Alan
    Tanabe, Yuichi
    Savolainen, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Mitochondrial DNA data indicate an introduction through Mainland Southeast Asia for Australian dingoes and Polynesian domestic dogs2012In: Proceedings of the Royal Society of London. Biological Sciences, ISSN 0962-8452, E-ISSN 1471-2954, Vol. 279, no 1730, p. 967-974Article in journal (Refereed)
    Abstract [en]

    In the late stages of the global dispersal of dogs, dingoes appear in the Australian archaeological record 3500 years BP, and dogs were one of three domesticates brought with the colonization of Polynesia, but the introduction routes to this region remain unknown. This also relates to questions about human history, such as to what extent the Polynesian culture was introduced with the Austronesian expansion from Taiwan or adopted en route, and whether pre-Neolithic Australia was culturally influenced by the surrounding Neolithic world. We investigate these questions by mapping the distribution of the mtDNA founder haplotypes for dingoes (A29) and ancient Polynesian dogs (Arc1 and Arc2) in samples across Southern East Asia (n = 424) and Island Southeast Asia (n = 219). All three haplotypes were found in South China, Mainland Southeast Asia and Indonesia but absent in Taiwan and the Philippines, and the mtDNA diversity among dingoes indicates an introduction to Australia 4600-18 300 years BP. These results suggest that Australian dingoes and Polynesian dogs originate from dogs introduced to Indonesia via Mainland Southeast Asia before the Neolithic, and not from Taiwan together with the Austronesian expansion. This underscores the complex origins of Polynesian culture and the isolation from Neolithic influence of the pre-Neolithic Australian culture.

  • 270. 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.

  • 271. Parasassi, T.
    et al.
    Brunelli, R.
    Bracci-Laudiero, L.
    Greco, G.
    Gustafsson, A. C.
    Krasnowska, E. K.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, T.
    Pittaluga, E.
    Romano, M. C.
    Serafino, A.
    Differentiation of normal and cancer cells induced by sulfhydryl reduction: biochemical and molecular mechanisms2005In: Cell Death and Differentiation, ISSN 1350-9047, E-ISSN 1476-5403, Vol. 12, no 10, p. 1285-1296Article in journal (Refereed)
    Abstract [en]

    We examined the morphological, biochemical and molecular outcome of a nonspecific sulfhydryl reduction in cells, obtained by supplementation of N-acetyl-L-cysteine (NAC) in a 0.1-10 mM concentration range. In human normal primary keratinocytes and in colon and ovary carcinoma cells we obtained evidences for: (i) a dose-dependent inhibition of proliferation without toxicity or apoptosis; (ii) a transition from a proliferative mesenchymal morphology to cell-specific differentiated structures; (iii) a noticeable increase in cell-cell and cell-substratum junctions; (iv) a relocation of the oncogenic beta-catenin at the cell-cell junctions; (v) inhibition of microtubules aggregation; (vi) upregulation of differentiation-related genes including p53, heat shock protein 27 gene, N-myc downstream-regulated gene 1, E-cadherin, and down-regulation of cyclooxygenase-2; (vii) inhibition of c-Src tyrosine kinase. In conclusion, a thiol reduction devoid of toxicity as that operated by NAC apparently leads to terminal differentiation of normal and cancer cells through a pleiade of converging mechanisms, many of which are targets of the recently developed differentiation therapy.

  • 272. Park, Christopher Y.
    et al.
    Klammer, Aaron A.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    MacCoss, Michael J.
    Noble, William S.
    Rapid and accurate peptide identification from tandem mass spectra2008In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 7, no 7, p. 3022-3027Article in journal (Refereed)
    Abstract [en]

    Mass spectrometry, the core technology in the field of proteomics, promises to enable scientists to identify and quantify the entire complement of proteins in a complex biological sample. Currently, the primary bottleneck in this type of experiment is computational. Existing algorithms for interpreting mass spectra are slow and fail to identify a large proportion of the given spectra. We describe a database search program called Crux that reimplements and extends the widely used database search program Sequest. For speed, Crux uses a peptide indexing scheme to rapidly retrieve candidate peptides for a given spectrum. For each peptide in the target database, Crux generates shuffled decoy peptides on the fly, providing a good null model and, hence, accurate false discovery rate estimates. Crux also implements two recently described postprocessing methods: a p value calculation based upon fitting a Weibull distribution to the observed scores, and a semisupervised method that learns to discriminate between target and decoy matches. Both methods significantly improve the overall rate of peptide identification. Crux is implemented in C and is distributed with source code freely to noncommercial users.

  • 273.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ståhl, Patrik L.
    A Novel Method for Rapid Hybridization of DNA to a Solid Support2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, p. e70504-Article in journal (Refereed)
    Abstract [en]

    Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself.

  • 274.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Generations of sequencing technologies2009In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 93, no 2, p. 105-111Article, review/survey (Refereed)
    Abstract [en]

    Advancements in the field of DNA sequencing are changing the scientific horizon and promising an era of personalized medicine for elevated human health. Although platforms are improving at the rate of Moore's Law, thereby reducing the sequencing costs by a factor of two or three each year, we find ourselves at a point in history where individual genomes are starting to appear but where the cost is still too high for routine sequencing of whole genomes. These needs will be met by miniaturized and parallelized platforms that allow a lower sample and template consumption thereby increasing speed and reducing costs. Current massively parallel, state-of-the-art systems are providing significantly improved throughput over Sanger systems and future single-molecule approaches will continue the exponential improvements in the field.

  • 275.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik L.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mahdessian, Hovsep
    KTH, School of Biotechnology (BIO), Gene Technology.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Visual DNA as a diagnostic tool2009In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 21, p. 3691-3695Article in journal (Refereed)
    Abstract [en]

    We report on the incorporation of the Visual DNA concept in a genotyping assay as a simple and straightforward detection tool. The principle of trapping streptavidin-coated superparamagnetic beads of micrometer size for visualization of genetic variances is used for PrASE-based detection of a panel of mutations in the severe and common genetic disorder of cystic fibrosis. The method allows a final investigation of genotypes by the naked eye and the output is easily documented using a regular hand-held device with an integrated digital camera. A number of samples were run through the assay, showing rapid and accurate detection using superparamagnetic beads and an off-the-shelf neodymium magnet. The assay emphasizes the power of Visual DNA and demonstrates the potential value of the method in future point-of-care tests.

  • 276.
    Pettersson, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Zajac, Pawel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Jacobsson, Josefin
    Fredriksson, Robert
    Marcus, Claude
    Schiöth, Helgi
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads2008In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 2, p. 323-329Article in journal (Refereed)
    Abstract [en]

    Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.

  • 277. Picelli, Simone
    et al.
    Zajac, Pawel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Zhou, Xiao-Lei
    Edler, David
    Lenander, Claes
    Dalen, Johan
    Hjern, Fredrik
    Lundqvist, Nils
    Lindforss, Ulrik
    Pahlman, Lars
    Smedh, Kennet
    Tornqvist, Anders
    Holm, Jorn
    Janson, Martin
    Andersson, Magnus
    Ekelund, Susanne
    Olsson, Louise
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lindblom, Annika
    Common variants in human CRC genes as low-risk alleles2010In: European Journal of Cancer, ISSN 0959-8049, E-ISSN 1879-0852, Vol. 46, no 6, p. 1041-1048Article in journal (Refereed)
    Abstract [en]

    The genetic susceptibility to colorectal cancer (CRC) has been estimated to be around 35% and yet high-penetrance germline mutations found so far explain less than 5% of all cases. Much of the remaining variations could be due to the co-inheritance of multiple low penetrant variants. The identification of all the susceptibility alleles could have public health relevance in the near future. To test the hypothesis that what are considered polymorphisms in human CRC genes could constitute low-risk alleles, we selected eight common SNPs for a pilot association study in 1785 cases and 1722 controls. One SNP, rs3219489:G>C (MUTYH Q324H) seemed to confer an increased risk of rectal cancer in homozygous status (OR = 1.52; CI = 1.06-2.17). When the analysis was restricted to our 'super-controls', healthy individuals with no family history for cancer, also rs1799977:A>G (MLH1 I219V) was associated with an increased risk in both colon and rectum patients with an odds ratio of 1.28 (CI = 1.02-1.60) and 1.34 (CI = 1.05-1.72), respectively (under the dominant model); while 2 SNPs, rs1800932:A>G (MSH6 P92P) and rs459552:T>A (APC D1822V) seemed to confer a protective effect. The latter, in particular showed an odds ratio of 0.76 (CI = 0.60-0.97) among colon patients and 0.73 (CI = 0.56-0.95) among rectal patients. In conclusion, our study suggests that common variants in human CRC genes could constitute low-risk alleles. (C) 2010 Elsevier Ltd. All rights reserved.

  • 278. 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.

  • 279. Quince, Christopher
    et al.
    Lundin, Elin E.
    Andreasson, Anna N.
    Greco, Dario
    Rafter, Joseph
    Talley, Nicholas J.
    Agreus, Lars
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Engstrand, Lars
    D'Amato, Mauro
    The impact of Crohn's disease genes on healthy human gut microbiota: a pilot study2013In: Gut, ISSN 0017-5749, E-ISSN 1468-3288, Vol. 62, no 6, p. 952-954Article in journal (Refereed)
  • 280.
    Redin, David
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Borgström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Institute (KI), Sweden.
    He, Mengxiao
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Aghelpasand, Hooman
    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.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Droplet Barcode Sequencing for targeted linked-read haplotyping of single DNA molecules2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 13, article id e125Article in journal (Refereed)
    Abstract [en]

    Data produced with short-read sequencing technologies result in ambiguous haplotyping and a limited capacity to investigate the full repertoire of biologically relevant forms of genetic variation. The notion of haplotype-resolved sequencing data has recently gained traction to reduce this unwanted ambiguity and enable exploration of other forms of genetic variation; beyond studies of just nucleotide polymorphisms, such as compound heterozygosity and structural variations. Here we describe Droplet Barcode Sequencing, a novel approach for creating linked-read sequencing libraries by uniquely barcoding the information within single DNA molecules in emulsion droplets, without the aid of specialty reagents or microfluidic devices. Barcode generation and template amplification is performed simultaneously in a single enzymatic reaction, greatly simplifying the workflow and minimizing assay costs compared to alternative approaches. The method has been applied to phase multiple loci targeting all exons of the highly variable Human Leukocyte Antigen A (HLA-A) gene, with DNA from eight individuals present in the same assay. Barcode-based clustering of sequencing reads confirmed analysis of over 2000 independently assayed template molecules, with an average of 753 reads in support of called polymorphisms. Our results show unequivocal characterization of all alleles present, validated by correspondence against confirmed HLA database entries and haplotyping results from previous studies.

  • 281. Regev, A
    et al.
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Danish Tech Univ, Denmark.
    Yosef, Nir
    et al.,
    The Human Cell Atlas2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e27041Article in journal (Refereed)
    Abstract [en]

    The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

  • 282.
    Reimegård, Johan
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Gene Technology.
    Kundu, Snehangshu
    Pendle, Ali
    Irish, Vivian F
    Shaw, Peter
    Nakayama, Naomi
    Sundström, Jens F
    Emanuelsson, Olof
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
    Abstract [en]

    Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction; out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation; DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation.

  • 283. Reynolds, Sheila M.
    et al.
    Käll, Lukas
    KTH, School of Biotechnology (BIO), Gene Technology.
    Riffle, Michael E.
    Bilmes, Jeff A.
    Noble, William Stafford
    Transmembrane topology and signal peptide prediction using dynamic bayesian networks2008In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 4, no 11, p. e1000213-Article in journal (Refereed)
    Abstract [en]

    Hidden Markov models (HMMs) have been successfully applied to the tasks of transmembrane protein topology prediction and signal peptide prediction. In this paper we expand upon this work by making use of the more powerful class of dynamic Bayesian networks (DBNs). Our model, Philius, is inspired by a previously published HMM, Phobius, and combines a signal peptide submodel with a transmembrane submodel. We introduce a two-stage DBN decoder that combines the power of posterior decoding with the grammar constraints of Viterbi-style decoding. Philius also provides protein type, segment, and topology confidence metrics to aid in the interpretation of the predictions. We report a relative improvement of 13% over Phobius in full-topology prediction accuracy on transmembrane proteins, and a sensitivity and specificity of 0.96 in detecting signal peptides. We also show that our confidence metrics correlate well with the observed precision. In addition, we have made predictions on all 6.3 million proteins in the Yeast Resource Center (YRC) database. This large-scale study provides an overall picture of the relative numbers of proteins that include a signal-peptide and/or one or more transmembrane segments as well as a valuable resource for the scientific community. All DBNs are implemented using the Graphical Models Toolkit. Source code for the models described here is available at http://noble.gs.washington.edu/proj/philius. A Philius Web server is available at http://www.yeastrc.org/philius, and the predictions on the YRC database are available at http://www.yeastrc.org/pdr.

  • 284. Ribacke, Ulf
    et al.
    Mok, Bobo W.
    Wirta, Valtteri
    Normark, Johan
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Kironde, Fred
    Egwang, Thomas G.
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics.
    Wahlgren, Mats
    Genome wide gene amplifications and deletions in Plasmodium falciparum2007In: Molecular and biochemical parasitology (Print), ISSN 0166-6851, E-ISSN 1872-9428, Vol. 155, no 1, p. 33-44Article in journal (Refereed)
    Abstract [en]

    The extent to which duplications and deletions occur in the Plasmodium falciparum genome, outside of the subtelomeres, and their contribution to the virulence of the malaria parasite is not known. Here we show the presence of multiple genome wide copy number polymorphisms (CNPs) covering 82 genes, the most extensive spanning a cumulative size of 110 kilobases. CNPs were identified in both laboratory strains and fresh clinical isolates using a 70-mer oligonucleotide microarray in conjunction with fluorescent in situ hybridizations and real-time quantitative PCR. The CNPs were found on all chromosomes except on chromosomes 6 and 8 and involved a total of 50 genes with increased copy numbers and 32 genes with decreased copy numbers relative to the 3D7 parasite. The genes, amplified in up to six copies, encode molecules involved in cell cycle regulation. cell division, drug resistance, erythrocyte invasion, sexual differentiation and unknown functions. These together with previous findings, suggest that the malaria parasite employs gene duplications and deletions as general strategies to enhance its survival and spread. Further analysis of the impact of discovered genetic differences and the underlying mechanisms is likely to generate a better understanding of the biology and the virulence of the malaria parasite.

  • 285. Robinson, Jonathan L.
    et al.
    Nielsen, Jens
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Integrative analysis of human omics data using biomolecular networks2016In: Molecular Biosystems, ISSN 1742-206X, E-ISSN 1742-2051, Vol. 12, no 10, p. 2953-2964Article, review/survey (Refereed)
    Abstract [en]

    High-throughput '-omics' technologies have given rise to an increasing abundance of genome-scale data detailing human biology at the molecular level. Although these datasets have already made substantial contributions to a more comprehensive understanding of human physiology and diseases, their interpretation becomes increasingly cryptic and nontrivial as they continue to expand in size and complexity. Systems biology networks offer a scaffold upon which omics data can be integrated, facilitating the extraction of new and physiologically relevant information from the data. Two of the most prevalent networks that have been used for such integrative analyses of omics data are genome-scale metabolic models (GEMs) and protein-protein interaction (PPI) networks, both of which have demonstrated success among many different omics and sample types. This integrative approach seeks to unite 'top-down' omics data with 'bottom-up' biological networks in a synergistic fashion that draws on the strengths of both strategies. As the volume and resolution of high-throughput omics data continue to grow, integrative network-based analyses are expected to play an increasingly important role in their interpretation.

  • 286. Rubin, Carl-Johan
    et al.
    Lindberg, Johan
    Fitzsimmons, Carolyn
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Gene Technology.
    Jensen, Per
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Andersson, Leif
    Kindmark, Andreas
    Differential gene expression in femoral bone from red junglefowl and domestic chicken, differing for bone phenotypic traits2007In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 8, p. 208-Article in journal (Refereed)
    Abstract [en]

    Background: Osteoporosis is frequently observed among aging hens from egg-producing strains (layers) of domestic chicken. White Leghorn (WL) has been intensively selected for egg production and it manifests striking phenotypic differences for a number of traits including several bone phenotypes in comparison with the wild ancestor of chicken, the red junglefowl (RJ). Previously, we have identified four Quantitative Trait Loci (QTL) affecting bone mineral density and bone strength in an intercross between RJ and WL. With the aim of further elucidating the genetic basis of bone traits in chicken, we have now utilized cDNA-microarray technology in order to compare global RNA-expression in femoral bone from adult RJ and WL (five of each sex and population). Results: When contrasting microarray data for all WL-individuals to that of all RJ-individuals we observed differential expression (False discovery rate adjusted p-values < 0.015) for 604 microarray probes. In corresponding male and female contrasts, differential expression was observed for 410 and 270 probes, respectively. Altogether, the three contrasts between WL and RJ revealed differential expression of 779 unique transcripts, 57 of which are located to previously identified QTL-regions for bone traits. Some differentially expressed genes have previously been attributed roles in bone metabolism and these were: WNT inhibitory factor I (WIFI), WD repeat-containing protein 5 (WDR5) and Syndecan 3 (SDC3). Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all 15 had lower expression in WL. Conclusion: We report the identification of 779 differentially expressed transcripts, several residing within QTL-regions for bone traits. Among differentially expressed transcripts, those encoding structural ribosomal proteins were highly enriched and all had lower expression levels in WL. In addition, transcripts encoding four translation initiation and translation elongation factor proteins also had lower expression levels in WL, possibly indicating perturbation of protein biosynthesis pathways between the two populations. Information derived from this study could be relevant to the bone research field and may also aid in further inference of genetic changes accompanying animal domestication.

  • 287.
    Ryden, Mikael
    et al.
    Karolinska Inst, Dept Med H7, S-14186 Stockholm, Sweden..
    Uzunel, Mehmet
    Karolinska Inst, Dept Clin Immunol, S-14186 Stockholm, Sweden..
    Hard, Joanna L.
    Karolinska Inst, Dept Cell & Mol Biol C5, S-17177 Stockholm, Sweden..
    Borgström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Mold, Jeff E.
    Karolinska Inst, Dept Cell & Mol Biol C5, S-17177 Stockholm, Sweden..
    Arner, Erik
    Karolinska Inst, Dept Med H7, S-14186 Stockholm, Sweden..
    Mejhert, Niklas
    Karolinska Inst, Dept Med H7, S-14186 Stockholm, Sweden..
    Andersson, Daniel P.
    Karolinska Inst, Dept Med H7, S-14186 Stockholm, Sweden..
    Widlund, Yvonne
    Karolinska Inst, Dept Med H7, S-14186 Stockholm, Sweden..
    Hassan, Moustapha
    Karolinska Inst, Dept Lab Med H5, S-14186 Stockholm, Sweden..
    Jones, Christina V.
    Karolinska Inst, Dept Cell & Mol Biol C5, S-17177 Stockholm, Sweden..
    Spalding, Kirsty L.
    Karolinska Inst, Dept Cell & Mol Biol C5, S-17177 Stockholm, Sweden..
    Svahn, Britt-Marie
    Karolinska Inst, Dept Clin Immunol, S-14186 Stockholm, Sweden..
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Frisen, Jonas
    Karolinska Inst, Dept Cell & Mol Biol C5, S-17177 Stockholm, Sweden..
    Bernard, Samuel
    Univ Lyon, CNRS UMR 5208, Inst Camille Jordan, F-69622 Villeurbanne, France..
    Mattsson, Jonas
    Karolinska Inst, Dept Clin Immunol, S-14186 Stockholm, Sweden..
    Arner, Peter
    Karolinska Inst, Dept Med H7, S-14186 Stockholm, Sweden..
    Transplanted Bone Marrow-Derived Cells Contribute to Human Adipogenesis2015In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 22, no 3, p. 408-417Article in journal (Refereed)
    Abstract [en]

    Because human white adipocytes display a high turnover throughout adulthood, a continuous supply of precursor cells is required to maintain adipogenesis. Bone marrow (BM)-derived progenitor cells may contribute to mammalian adipogenesis; however, results in animal models are conflicting. Here we demonstrate in 65 subjects who underwent allogeneic BM or peripheral blood stem cell (PBSC) transplantation that, over the entire lifespan, BM/PBSC-derived progenitor cells contribute similar to 10% to the subcutaneous adipocyte population. While this is independent of gender, age, and different transplantation-related parameters, body fat mass exerts a strong influence, with up to 2.5-fold increased donor cell contribution in obese individuals. Exome and whole-genome sequencing of single adipocytes suggests that BM/PBSC-derived progenitors contribute to adipose tissue via both differentiation and cell fusion. Thus, at least in the setting of transplantation, BM serves as a reservoir for adipocyte progenitors, particularly in obese subjects.

  • 288.
    Sahlin, Kristoffer
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Street, Nathaniel
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Arvestad, Lars
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Improved gap size estimation for scaffolding algorithms2012In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 28, no 17, p. 2215-2222Article in journal (Refereed)
    Abstract [en]

    Motivation: One of the important steps of genome assembly is scaffolding, in which contigs are linked using information from read-pairs. Scaffolding provides estimates about the order, relative orientation and distance between contigs. We have found that contig distance estimates are generally strongly biased and based on false assumptions. Since erroneous distance estimates can mislead in subsequent analysis, it is important to provide unbiased estimation of contig distance.Results: In this article, we show that state-of-the-art programs for scaffolding are using an incorrect model of gap size estimation. We discuss why current maximum likelihood estimators are biased and describe what different cases of bias we are facing. Furthermore, we provide a model for the distribution of reads that span a gap and derive the maximum likelihood equation for the gap length. We motivate why this estimate is sound and show empirically that it outperforms gap estimators in popular scaffolding programs. Our results have consequences both for scaffolding software, structural variation detection and for library insert-size estimation as is commonly performed by read aligners.

  • 289.
    Sahlin, Kristoffer
    et al.
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vezzi, Francesco
    KTH, School of Computer Science and Communication (CSC), Computational Biology, CB. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystedt, Björn
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Arvestad, Lars
    BESST - Efficient scaffolding of large fragmented assemblies2014In: BMC Bioinformatics, ISSN 1471-2105, E-ISSN 1471-2105, Vol. 15, no 1, p. 281-Article in journal (Refereed)
    Abstract [en]

    Background: The use of short reads from High Throughput Sequencing (HTS) techniques is now commonplace in de novo assembly. Yet, obtaining contiguous assemblies from short reads is challenging, thus making scaffolding an important step in the assembly pipeline. Different algorithms have been proposed but many of them use the number of read pairs supporting a linking of two contigs as an indicator of reliability. This reasoning is intuitive, but fails to account for variation in link count due to contig features. We have also noted that published scaffolders are only evaluated on small datasets using output from only one assembler. Two issues arise from this. Firstly, some of the available tools are not well suited for complex genomes. Secondly, these evaluations provide little support for inferring a software's general performance. Results: We propose a new algorithm, implemented in a tool called BESST, which can scaffold genomes of all sizes and complexities and was used to scaffold the genome of P. abies (20 Gbp). We performed a comprehensive comparison of BESST against the most popular stand-alone scaffolders on a large variety of datasets. Our results confirm that some of the popular scaffolders are not practical to run on complex datasets. Furthermore, no single stand-alone scaffolder outperforms the others on all datasets. However, BESST fares favorably to the other tested scaffolders on GAGE datasets and, moreover, outperforms the other methods when library insert size distribution is wide. Conclusion: We conclude from our results that information sources other than the quantity of links, as is commonly used, can provide useful information about genome structure when scaffolding.

  • 290.
    Sahlén, Pelin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Abdullayev, Ilgar
    Ramsköld, Daniel
    Matskova, Liudmila
    Rilakovic, Nemanja
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lötstedt, Britta
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Albert, Thomas J.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Sandberg, Rickard
    Genome-wide mapping of promoter-anchored interactions with close to single-enhancer resolution2015In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 16, article id 156Article in journal (Refereed)
    Abstract [en]

    Although the locations of promoters and enhancers have been identified in several cell types, we still have limited information on their connectivity. We developed HiCap, which combines a 4-cutter restriction enzyme Hi-C with sequence capture of promoter regions. Applying the method to mouse embryonic stem cells, we identified promoter-anchored interactions involving 15,905 promoters and 71,984 distal regions. The distal regions were enriched for enhancer marks and transcription, and had a mean fragment size of only 699 bp - close to single-enhancer resolution. High-resolution maps of promoter-anchored interactions with HiCap will be important for detailed characterizations of chromatin interaction landscapes.

  • 291.
    Salmén, Fredrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Spatially resolved and single cell transcriptomics2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In recent years, massive parallel sequencing has revolutionized the field of biology and has provided us with a vast number of new discoveries in fields such as neurology, developmental biology and cancer research. A significant area is deciphering gene expression patterns, as well as other aspects of transcriptome information, such as the impact of splice variants and mutations on biological functions and disease development. By applying RNA-sequencing, one can extract this type of information in a large-scale manner. The most recent approaches include high-resolution techniques such as single cell sequencing and in situ methods in order to circumvent the problems with gene expression averaging in homogenized samples, and loss of spatial information.

    The research in this thesis is focused on the development of a novel genome-wide spatial transcriptomics method. The technique is used for analysis of intact tissue sections as well as single cells from solution, with the aim to combine gene expression and morphological information. In Paper I, the method is described in detail, and it is shown that the method is able to generate spatial high quality data from mouse olfactory bulb tissue sections (a part of the forebrain) as well as from tissue sections from breast cancer samples. In Paper III, we adapt the library preparation method in order to be able to execute it on a robotic workstation, thus increasing the reproducibility and the throughput, and decreasing the hands-on time. In Paper IV, we generate 3D-data from breast cancer samples by serial sectioning. We show that the gene expression can be highly variable along all three axes of a tumor, and we track pathways with specific spatial activity, as well as perform subtype classification with three-dimensional resolution. In Paper II, we present a high-throughput method for single cell transcriptomics of cells in solution. The method is based on the same type of solid surface capture as the tissue protocol described in Papers I, III and IV. Again, we show that we can generate high-quality gene expression data, and connect this to morphological characteristics of the analyzed single cells; both using cultured cells and samples from patients with leukemia.

  • 292.
    Salmén, Fredrik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Vickovic, Sanja
    KTH, School of Biotechnology (BIO), Gene Technology.
    Stenbeck, Linnea
    KTH, School of Biotechnology (BIO), Gene Technology.
    Vallon-Christersson, Johan
    Lund.
    Ehinger, Anna
    Häkkinen, Jari
    Borg, Åke
    Frisén, Jonas
    Ståhl, Patrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Three-dimensional spatial transcriptomics analysis for classification of breast cancerManuscript (preprint) (Other academic)
  • 293. Salvo, P.
    et al.
    Henry, O. Y. F.
    Dhaenens, K.
    Acero Sanchez, J. L.
    Gielen, A.
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    O'Sullivan, C. K.
    Vanfleteren, J.
    Fabrication and functionalization of PCB gold electrodes suitable for DNA-based electrochemical sensing2014In: Bio-medical materials and engineering, ISSN 0959-2989, E-ISSN 1878-3619, Vol. 24, no 4, p. 1705-1714Article in journal (Refereed)
    Abstract [en]

    The request of high specificity and selectivity sensors suitable for mass production is a constant demand in medical research. For applications in point-of-care diagnostics and therapy, there is a high demand for low cost and rapid sensing platforms. This paper describes the fabrication and functionalization of gold electrodes arrays for the detection of deoxyribonucleic acid (DNA) in printed circuit board (PCB) technology. The process can be implemented to produce efficiently a large number of biosensors. We report an electrolytic plating procedure to fabricate low-density gold microarrays on PCB suitable for electrochemical DNA detection in research fields such as cancer diagnostics or pharmacogenetics, where biosensors are usually targeted to detect a small number of genes. PCB technology allows producing high precision, fast and low cost microelectrodes. The surface of the microarray is functionalized with self-assembled monolayers of mercaptoundodecanoic acid or thiolated DNA. The PCB microarray is tested by cyclic voltammetry in presence of 5 mM of the redox probe K3Fe(CN6) in 0.1 M KCl. The voltammograms prove the correct immobilization of both the alkanethiol systems. The sensor is tested for detecting relevant markers for breast cancer. Results for 5 nM of the target TACSTD1 against the complementary TACSTD1 and non-complementary GRP, MYC, SCGB2A1, SCGB2A2, TOP2A probes show a remarkable detection limit of 0.05 nM and a high specificity.

  • 294. Sandalova, T.
    et al.
    Michaelsson, J.
    Harris, R. A.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    Schneider, G.
    Karres, K.
    Achour, A.
    A structural basis for CD8(+) T cell-dependent recognition of non-homologous peptide ligands - Implications for molecular mimicry in autoreactivity2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 29, p. 27069-27075Article in journal (Refereed)
    Abstract [en]

    Molecular mimicry of self-epitopes by viral antigens is one possible pathogenic mechanism underlying induction of autoimmunity. A self-epitope, mDBM, derived from mouse dopamine beta-mono-oxygenase (KALYDYAPI) sharing 44% sequence identity with the lymphocytic choriomeningitis virus-derived immunodominant epitope gp33 (KAVYNFATC/ M), has previously been identified as a cross-reactive self-ligand, presentation of which results in autoimmunity. A rat peptide homologue, rDBM (KALYNYAPI, 56% identity to gp33), which displayed similar properties to mDBM, has also been identified. We herein report the crystal structure of H-2D(b)center dot rDBM and a comparison with the crystal structures of the cross-reactive H-2D(b)center dot gp33 and non- cross- reactive H-2D(b)center dot gp33 (V3L) escape variant (KALYNFATM, 88% identity to gp33). Despite the large sequence disparity, rDBM and gp33 peptides are presented in nearly identical manners by H-2D(b), with a striking juxtaposition of the central sections of both peptides from residues p3 to p7. The structural similarity provides H-2D(b) in complex with either a virus-derived or a dopamine beta-mono-oxygenase-derived peptide with a shared antigenic identity that conserves the positioning of the heavy chain and peptide residues that interact with the T cell receptor (TCR). This stands in contrast to the structure of H-2D(b) center dot gp33 (V3L), in which a single conserved mutation, also present in rDBM, induces large movements of both the peptide backbone and the side chains that interact with the TCR. The TCR-interacting surfaces of the H-2D(b) center dot rDBM and H-2D(b) center dot gp33 major histocompatibility complexes are very similar with regard to shape, topology, and charge distribution, providing a structural basis for CD8 T cell activation by molecular mimicry and potential subsequent development of autoreactivity.

  • 295.
    Sandberg, Julia
    KTH, School of Biotechnology (BIO), Gene Technology.
    Massively parallel analysis of cells and nucleic acids2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Recent proceedings in biotechnology have enabled completely new avenues in life science research to be explored. By allowing increased parallelization an ever-increasing complexity of cell samples or experiments can be investigated in shorter time and at a lower cost. This facilitates for example large-scale efforts to study cell heterogeneity at the single cell level, by analyzing cells in parallel that also can include global genomic analyses. The work presented in this thesis focuses on massively parallel analysis of cells or nucleic acid samples, demonstrating technology developments in the field as well as use of the technology in life sciences.

    In stem cell research issues such as cell morphology, cell differentiation and effects of reprogramming factors are frequently studied, and to obtain information on cell heterogeneity these experiments are preferably carried out on single cells. In paper I we used a high-density microwell device in silicon and glass for culturing and screening of stem cells. Maintained pluripotency in stem cells from human and mouse was demonstrated in a screening assay by antibody staining and the chip was furthermore used for studying neural differentiation. The chip format allows for low sample volumes and rapid high-throughput analysis of single cells, and is compatible with Fluorescence Activated Cell Sorting (FACS) for precise cell selection.

    Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences by constantly producing increasing amounts of data from one sequencing run. However, the reagent costs and labor requirements in current massively parallel sequencing protocols are still substantial. In paper II-IV we have focused on flow-sorting techniques for improved sample preparation in bead-based massive sequencing platforms, with the aim of increasing the amount of quality data output, as demonstrated on the Roche/454 platform. In paper II we demonstrate a rapid alternative to the existing shotgun sample titration protocol and also use flow-sorting to enrich for beads that carry amplified template DNA after emulsion PCR, thus obtaining pure samples and with no downstream sacrifice of DNA sequencing quality. This should be seen in comparison to the standard 454-enrichment protocol, which gives rise to varying degrees of sample purity, thus affecting the sequence data output of the sequencing run. Massively parallel sequencing is also useful for deep sequencing of specific PCR-amplified targets in parallel. However, unspecific product formation is a common problem in amplicon sequencing and since these shorter products may be difficult to fully remove by standard procedures such as gel purification, and their presence inevitably reduces the number of target sequence reads that can be obtained in each sequencing run. In paper III a gene-specific fluorescent probe was used for target-specific FACS enrichment to specifically enrich for beads with an amplified target gene on the surface. Through this procedure a nearly three-fold increase in fraction of informative sequences was obtained and with no sequence bias introduced. Barcode labeling of different DNA libraries prior to pooling and emulsion PCR is standard procedure to maximize the number of experiments that can be run in one sequencing lane, while also decreasing the impact of technical noise. However, variation between libraries in quality and GC content affects amplification efficiency, which may result in biased fractions of the different libraries in the sequencing data. In paper IV barcode specific labeling and flow-sorting for normalization of beads with different barcodes on the surface was used in order to weigh the proportion of data obtained from different samples, while also removing mixed beads, and beads with no or poorly amplified product on the surface, hence also resulting in an increased sequence quality.

    In paper V, cell heterogeneity within a human being is being investigated by low-coverage whole genome sequencing of single cell material. By focusing on the most variable portion of the human genome, polyguanine nucleotide repeat regions, variability between different cells is investigated and highly variable polyguanine repeat loci are identified. By selectively amplifying and sequencing polyguanine nucleotide repeats from single cells for which the phylogenetic relationship is known, we demonstrate that massively parallel sequencing can be used to study cell-cell variation in length of these repeats, based on which a phylogenetic tree can be drawn.

  • 296.
    Sandberg, Julia
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Borgström, Erik
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ernst, Aurélie
    Paterlini, Maria
    Réu, Pedro
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Frisén, Jonas
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Interrogation of polyguaninine nucleotide repeat variability in human T-cells by whole genome sequencing of single cellsManuscript (preprint) (Other academic)
    Abstract [en]

    Polyguanine nucleotide repeats exhibit a greater degree of variation than the average for the genome as a whole. This is partly due to polymerase slippage that causes insertions or deletions in these repeat sequence. The high variability of these repeats makes them useful for tracking the differentiation and fate of cells within tissues and organs. However, the same factors that create this variability also give rise to technical difficulties in DNA amplification and massively parallel DNA sequencing. In the study reported herein, we investigated shotgun sequence data from a standard multi-cell sample as well as sequence data for four single cells from the same individual. This was used to assess sequence quality in whole genome amplified single cell material and to investigate variability in homopolymeric regions between individual T-cells. In a more focused study, a selected set of polyG loci in single cells for which the phylogenetic relationship was known, were amplified and sequence determined. Based on the length differences in polyG repeats between the eight cells a phylogenetic tree was constructed, that was very similar to the known tree.

  • 297.
    Sandberg, Julia
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Neiman, Mårten
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Gene-specific FACS sorting method for target selection in high-throughput amplicon sequencing2010In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 11, no 140Article in journal (Refereed)
    Abstract [en]

    Background

    In addition to shotgun sequencing, next generation sequencing has been shown to be suitable for deep sequencing of many specific PCR-amplified target genes in parallel. However, unspecific product formation is a common problem in amplicon sequencing since these fragments are difficult to fully remove by gel purification, and their presence inevitably reduces the number of mappable sequence reads that can be obtained in each sequencing run.

    Results

    We have used a novel flow cytometric sorting approach to specifically enrich Roche/454 DNA Capture beads carrying target DNA sequences on their surface, and reject beads carrying unspecific sequences. This procedure gives a nearly three-fold increase in the fraction of informative sequences obtained. Presented results also show that there are no significant differences in the distribution or presence of different genotypes between a FACS-enriched sample and a standard-enriched control sample.

    Conclusions

    Target-specific FACS enrichment prior to Roche/454 sequencing provides a quick, inexpensive way of increasing the amount of high quality data obtained in a single sequencing run, without introducing any sequence bias.

  • 298.
    Sandberg, Julia
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ståhl, Patrik L.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Ahmadian, Afshin
    KTH, School of Biotechnology (BIO), Gene Technology.
    Bjursell, Magnus K
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Flow cytometry for enrichment and titration in massively parallel DNA sequencing2009In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 8Article in journal (Refereed)
    Abstract [en]

    Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols.

  • 299.
    Sandberg, Julia
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Werne, Beata
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Dessing, Mark
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rapid flow-sorting to simultaneously resolve multiplex massively parallel sequencing products2011In: Scientific Reports, ISSN 2045-2322, Vol. 1, no 108Article in journal (Refereed)
    Abstract [en]

    Sample preparation for Roche/454, ABI/SOLiD and Life Technologies/Ion Torrent sequencing are based on amplification of library fragments on the surface of beads prior to sequencing. Commonly, libraries are barcoded and pooled, to maximise the sequence output of each sequence run. Here, we describe a novel approach for normalization of multiplex next generation sequencing libraries after emulsion PCR. Briefly, amplified libraries carrying unique barcodes are prepared by fluorescent tagging of complementary sequences and then resolved by high-speed flow cytometric sorting of labeled emulsion PCR beads. The protocol is simple and provides an even sequence distribution of multiplex libraries when sequencing the flow-sorted beads. Moreover, since many empty and mixed emulsion PCR beads are removed, the approach gives rise to a substantial increase in sequence quality and mean read length, as compared to that obtained by standard enrichment protocols.

  • 300.
    Sandholm, Thomas
    et al.
    KTH, School of Computer Science and Communication (CSC), Centres, Centre for High Performance Computing, PDC. KTH, School of Information and Communication Technology (ICT), Computer and Systems Sciences, DSV.
    Lai, Kevin
    Hewlett-Packard Laboratories, Information Dynamics Laboratory, Palo Alto.
    Andrade, Jorge
    KTH, School of Biotechnology (BIO), Gene Technology.
    Odeberg, Jacob
    KTH, School of Biotechnology (BIO), Gene Technology.
    Market-Based Resource Allocation using Price Prediction in a high performance computing Grid for scientific applications2006In: Proceedings of the IEEE International Symposium on High Performance Distributed Computing 2006, 2006, Vol. 15th IEEE International Symposium, p. 132-143Conference paper (Refereed)
    Abstract [en]

    We present the implementation and analysis of a market-based resource allocation system for computational Grids. Although Grids provide a way to share resources and take advantage of statistical multiplexing, a variety of challenges remain. One is the economically efficient allocation of resources to users from disparate organizations who have their own and sometimes conflicting requirements for both the quantity and quality of services. Another is secure and scalable authorization despite rapidly changing allocations.

    Our solution to both of these challenges is to use a market-based resource allocation system. This system allows users to express diverse quantity- and quality-of-service requirements, yet prevents them from denying service to other users. It does this by providing tools to the user to predict and tradeoff risk and expected return in the computational market. In addition, the system enables secure and scalable authorization by using signed money-transfer tokens instead of identity-based authorization. This removes the overhead of maintaining and updating access control lists, while restricting usage based on the amount of money transferred We examine the performance of the system by running a bioinformatics application on a fully operational implementation of an integrated Grid market.

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