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  • 1.
    Akan, Pelin
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Alexeyenko, Andrey
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Costea, Paul Igor
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hedberg, Lilia
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Werne Solnestam, Beata
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallman, Jimmie
    Lundberg, Emma
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines2012In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 4, p. 86-Article in journal (Refereed)
    Abstract [en]

    We here present a comparative genome, transcriptome and functional network analysis of three human cancer cell lines (A431, U251MG and U2OS), and investigate their relation to protein expression. Gene copy numbers significantly influenced corresponding transcript levels; their effect on protein levels was less pronounced. We focused on genes with altered mRNA and/or protein levels to identify those active in tumor maintenance. We provide comprehensive information for the three genomes and demonstrate the advantage of integrative analysis for identifying tumor-related genes amidst numerous background mutations by relating genomic variation to expression/protein abundance data and use gene networks to reveal implicated pathways.

  • 2. Ameur, Adam
    et al.
    Dahlberg, Johan
    Olason, Pall
    Vezzi, Francesco
    Karlsson, Robert
    Martin, Marcel
    Viklund, Johan
    Kahari, Andreas Kusalananda
    Lundin, Par
    Che, Huiwen
    Thutkawkorapin, Jessada
    Eisfeldt, Jesper
    Lampa, Samuel
    Dahlberg, Mats
    Hagberg, Jonas
    Jareborg, Niclas
    Liljedahl, Ulrika
    Jonasson, Inger
    Johansson, Asa
    Feuk, Lars
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Syvanen, Ann-Christine
    Lundin, Sverker
    Nilsson, Daniel
    Nystedt, Bjorn
    Magnusson, Patrik K. E.
    Gyllensten, Ulf
    SweGen: a whole-genome data resource of genetic variability in a cross-section of the Swedish population2017In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 25, no 11, p. 1253-1260Article in journal (Refereed)
    Abstract [en]

    Here we describe the SweGen data set, a comprehensive map of genetic variation in the Swedish population. These data represent a basic resource for clinical genetics laboratories as well as for sequencing-based association studies by providing information on genetic variant frequencies in a cohort that is well matched to national patient cohorts. To select samples for this study, we first examined the genetic structure of the Swedish population using high-density SNP-array data from a nation-wide cohort of over 10 000 Swedish-born individuals included in the Swedish Twin Registry. A total of 1000 individuals, reflecting a cross-section of the population and capturing the main genetic structure, were selected for whole-genome sequencing. Analysis pipelines were developed for automated alignment, variant calling and quality control of the sequencing data. This resulted in a genome-wide collection of aggregated variant frequencies in the Swedish population that we have made available to the scientific community through the website https://swefreq.nbis.se. A total of 29.2 million single-nucleotide variants and 3.8 million indels were detected in the 1000 samples, with 9.9 million of these variants not present in current databases. Each sample contributed with an average of 7199 individual-specific variants. In addition, an average of 8645 larger structural variants (SVs) were detected per individual, and we demonstrate that the population frequencies of these SVs can be used for efficient filtering analyses. Finally, our results show that the genetic diversity within Sweden is substantial compared with the diversity among continental European populations, underscoring the relevance of establishing a local reference data set.

  • 3.
    Borgström, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Large Scale Library Generation for High Throughput Sequencing Authors and Affiliations2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 4, p. e19119-Article in journal (Refereed)
    Abstract [en]

    Background: Large efforts have recently been made to automatethe sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols. Methodology/Principal Findings: In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions. Conclusions/Significance: The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.

  • 4.
    Borgström, Erik
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Redin, David
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Berglund, Emelie
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Andersson, Anders F.
    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.
    Phasing of single DNA molecules by massively parallel barcoding2015In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, article id 7173Article in journal (Refereed)
    Abstract [en]

    High-throughput sequencing platforms mainly produce short-read data, resulting in a loss of phasing information for many of the genetic variants analysed. For certain applications, it is vital to know which variant alleles are connected to each individual DNA molecule. Here we demonstrate a method for massively parallel barcoding and phasing of single DNA molecules. First, a primer library with millions of uniquely barcoded beads is generated. When compartmentalized with single DNA molecules, the beads can be used to amplify and tag any target sequences of interest, enabling coupling of the biological information from multiple loci. We apply the assay to bacterial 16S sequencing and up to 94% of the hypothesized phasing events are shown to originate from single molecules. The method enables use of widely available short-read-sequencing platforms to study long single molecules within a complex sample, without losing phase information.

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

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

  • 6.
    Hugerth, Luisa W.
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Wefer, Hugo A.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jakobsson, Hedvig E.
    Lindberg, Mathilda
    Rodin, Sandra
    Engstrand, Lars
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    DegePrime, a Program for Degenerate Primer Design for Broad-Taxonomic-Range PCR in Microbial Ecology Studies2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 16, p. 5116-5123Article in journal (Refereed)
    Abstract [en]

    The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e. g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.

  • 7.
    Lundin, Sverker
    KTH, School of Biotechnology (BIO), Gene Technology.
    Methods to Prepare DNA for Efficient Massive Sequencing2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity.

    The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding.

    Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments.

  • 8.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gruselius, Joel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystedt, Björn
    Science for Life Laboratory, Stockholm University, Department of Biochemistry and Biophysics, Stockholm.
    Lexow, Preben
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, p. 1186-Article in journal (Refereed)
    Abstract [en]

    Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.

  • 9.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jemt, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Finn, Terje-Hegge
    Foam, Napoleon
    Pettersson, Erik
    Käller, Max
    Wirta, Valtteri
    Lexow, Preben
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Endonuclease specificity and sequence dependence of Type IIS restriction enzymes2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 1, article id e0117059Article in journal (Refereed)
    Abstract [en]

    Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

  • 10.
    Lundin, Sverker
    et al.
    KTH, School of Biotechnology (BIO), Gene Technology.
    Stranneheim, Henrik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Pettersson, Erik
    KTH, School of Biotechnology (BIO), Gene Technology.
    Klevebring, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology.
    Increased Throughput by Parallelization of Library Preparation for Massive Sequencing2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 3, p. e10029-Article in journal (Refereed)
    Abstract [en]

    Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.

  • 11.
    Neiman, Mårten
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Savolainen, Peter
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ahmadian, Afshin
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Decoding a substantial set of samples in parallel by massive sequencing2011In: Plos One, ISSN 1932-6203, Vol. 6, no 3Article in journal (Refereed)
    Abstract [en]

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

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

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