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  • 1.
    Borgström, Erik
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Redin, David
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Lundin, Sverker
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Berglund, Emelie
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Andersson, Anders F.
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Phasing of single DNA molecules by massively parallel barcoding2015Inngår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 6, artikkel-id 7173Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 2.
    Dezfouli, Mahya
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Redin, David
    KTH, Skolan för bioteknologi (BIO), Proteinteknologi.
    Borgström, Erik
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Edfors, Fredrik
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Uhlén, Mathias
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Schwenk, Jochen M.
    KTH, Skolan för bioteknologi (BIO), Proteomik och nanobioteknologi.
    Ahmadian, Afshin
    KTH, Skolan för bioteknologi (BIO), Genteknologi.
    Droplet-based Immuno-Sequencing to Deconvolute Affinity Recognition EventsManuskript (preprint) (Annet vitenskapelig)
  • 3.
    Johansson, Sebastian
    et al.
    Stockholms Universitet.
    Juhos, Szilveszter
    Stockholms Universitet.
    Redin, David
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Ahmadian, Afshin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Käller, Max
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Comprehensive haplotyping of the HLA gene family using nanopore sequencingManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The HLA gene family is the most polymorphic loci in the human genome; it encodes for the major histocompatibility complexes (MHC) which mediates the immune response in terms of cellular interactions with antigens. Compatibility between HLA alleles is thus of great medical interest for recipients of allogeneic transplantations. Traditional serological techniques to evaluate compatibility are now being replaced by more accurate DNA sequencing-based methods. However, short read sequencing data typically result in collapsed sequences representing a mixture of variants from native haplotypes. In addition, most previous studies have been limited to a few highly polymorphic exons of various HLA genes. Here we present haplotype-resolved full-length sequencing of the six most clinically relevant MHC Class I and Class II genes, to characterize the haplotypes of eight reference individuals, using a single MinION flow cell. The results show that full-length sequencing of single molecules enables haplotypes to be resolved to the highest degree of accuracy (four-field resolution). In this study, a majority of the alleles were classified with four-field resolution and could be verified through previously published genotyping studies. These results support the notion that nanopore sequencing could be a viable solution for highly accurate clinical evaluation of histocompatibility.

  • 4.
    Redin, David
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Phasing single DNA molecules with barcode linked sequencing2018Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Elucidation of our genetic constituents has in the past decade predominately taken the form of short-read DNA sequencing. Revolutionary technology developments have enabled vast amounts of biological information to be obtained, but from a medical standpoint it has yet to live up to the promise of associating individual genotypes to phenotypic states of wide-spread clinical relevance. The mechanisms by which complex phenotypes arise have been difficult to ascertain and the value of short-read sequencing platforms have been limited in this regard. It has become evident that resolving the full spectrum of genetic heterogeneity requires accurate long range information of individual haplotypes to be distinguished. Long-range haplotyping information can be obtained experimentally by long-read sequencing platforms or through linkage of short sequencing reads by means of a common barcode. This thesis explores these solutions, primarily through the development of novel technologies to phase short sequences of single molecules using DNA barcoding. A new method for high-throughput phasing of single DNA molecules, achieved by the production and utilization of uniquely barcoded beads in emulsion droplets, is described in Paper I. The results confirm that complex libraries of beads featuring mutually exclusive barcodes can be generated through clonal PCR amplification, and that these beads can be used to phase variations of the 16s rRNA gene which reduces the ambiguity of classifying bacterial species for metagenomics. Paper II describes a second methodology (‘Droplet Barcode Sequencing’) which simplifies the concept of barcoding DNA fragments by omitting the need for beads and instead relying on clonal amplification of single barcoding oligonucleotides. This study also increases the amount of information that can be linked, which is showcased by phasing all exons of the HLA-A gene and successfully resolving all the alleles present in a sample pool of eight individuals. Paper III expands on this work and explores the use of a single molecule sequencing platform to provide full-length sequencing coverage of six genes of the HLA family. The results show that while genes shorter than 10 kb can be resolved with a high degree of accuracy, compensating for a relatively high error rate by means of increased coverage can be challenging for larger genomic loci. Finally, Paper IV introduces the use of barcode-linked reads on an unprecedented scale, with a new assay that enables low-cost haplotyping of whole genomes without the need for predetermined capture sequences. This technology is utilized to generate a haplotype-resolved human genome, call large-scale structural variants and perform reference-free assembly of bacterial and human genomes. At a cost of only $19 USD per sample, this technology makes the benefits of long-range haplotyping available to the vast majority of laboratories which currently rely solely on short-read sequencing platforms.

  • 5.
    Redin, David
    et al.
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Borgström, Erik
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab. Karolinska Institute (KI), Sweden.
    He, Mengxiao
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Aghelpasand, Hooman
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, Skolan för bioteknologi (BIO), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Droplet Barcode Sequencing for targeted linked-read haplotyping of single DNA molecules2017Inngår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, nr 13, artikkel-id e125Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 6.
    Redin, David
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Frick, Tobias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Aghelpasand, Hooman
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Borgström, Erik
    KTH, Centra, Science for Life Laboratory, SciLifeLab. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Olsen, Remi-Andre
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, Box 1031, S-17121 Solna, Sweden..
    Ahmadian, Afshin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    High throughput barcoding method for genome-scale phasing2019Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikkel-id 18116Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a novel library preparation method for high throughput barcoding of short reads where millions of random barcodes can be used to reconstruct megabase-scale phase blocks.

  • 7.
    Redin, David
    et al.
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Frick, Tobias
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Aghelpasand, Hooman
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Theland, Jennifer
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Käller, Max
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Borgström, Erik
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Olsen, Remi-Andre
    Stockholms Universitet.
    Ahmadian, Afshin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi.
    Efficient whole genome haplotyping and single molecule phasing with barcode-linked readsManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a library preparation method which enables whole genome haplotyping, long-range phasing of single DNA molecules, and de novo genome assembly through barcode-linked reads (BLR). Millions of random barcodes are used to reconstruct megabase-scale phase blocks and call structural variants. We also highlight the versatility of our technology by generating libraries from different organisms using picograms to nanograms of input material.

  • 8.
    Zhang, Miao
    et al.
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Material- och nanofysik. KTH.
    Ngampeerapong, Chonmanart
    Redin, David
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Ahmadian, Afshin
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Genteknologi. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Sychugov, Ilya
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Material- och nanofysik.
    Linnros, Jan
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Material- och nanofysik.
    Thermophoresis-Controlled Size-Dependent DNA Translocation through an Array of Nanopores2018Inngår i: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 12, nr 5, s. 4574-4582Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Large arrays of nanopores can be used for high-throughput biomolecule translocation with applications toward size discrimination and sorting at the single-molecule level. In this paper, we propose to discriminate DNA length by the capture rate of the molecules to an array of relatively large nanopores (50–130 nm) by introducing a thermal gradient by laser illumination in front of the pores balancing the force from an external electric field. Nanopore arrays defined by photolithography were batch processed using standard silicon technology in combination with electrochemical etching. Parallel translocation of single, fluorophore-labeled dsDNA strands is recorded by imaging the array with a fast CMOS camera. The experimental data show that the capture rates of DNA molecules decrease with increasing DNA length due to the thermophoretic effect of the molecules. It is shown that the translocation can be completely turned off for the longer molecule using an appropriate bias, thus allowing a size discrimination of the DNA translocation through the nanopores. A derived analytical model correctly predicts the observed capture rate. Our results demonstrate that by combining a thermal and a potential gradient at the nanopores, such large nanopore arrays can potentially be used as a low-cost, high-throughput platform for molecule sensing and sorting.

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