| 1. |
- Gharizadeh, Baback, et al.
(författare)
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Methodological improvements of pyrosequencing technology
- 2006
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 124:3, s. 504-511
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Tidskriftsartikel (refereegranskat)abstract
- Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.
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| 2. |
- Akhras, Michael, et al.
(författare)
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Connector Inversion Probe Technology : A Powerful One- Primer Multiplex DNA Amplification System for Numerous Scientific Applications
- 2007
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:9, s. e915-
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Tidskriftsartikel (refereegranskat)abstract
- We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i. e. "multiplex multiplexing padlocks'' (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs.
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| 3. |
- Akhras, Michael, et al.
(författare)
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PathogenMip Assay : A Multiplex Pathogen Detection Assay
- 2007
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 2:2, s. e223-
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Tidskriftsartikel (refereegranskat)abstract
- The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe.
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| 4. |
- Akhras, Michael S., 1980-
(författare)
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Nucleic Acid Based Pathogen Diagnostics
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pathogenic organisms are transmitted to the host organism through all possible connected pathways, and cause a myriad of diseases states. Commonly occurring curable infectious diseases still impose the greatest health impacts on a worldwide perspective. The Bill & Melinda Gates Foundation partnered with RAND Corporation to form the Global Health Diagnostics Forum, with the goal of establishing and interpreting mathematical models for what effects a newly introduced point-of-care pathogen diagnostic would have in developing countries. The results were astonishing, with potentially millions of lives to be saved on an annual basis. Golden standard for diagnostics of pathogenic bacteria has long been cultureable medias. Environmental biologists have estimated that less than 1% of all bacteria are cultureable. Genomic-based approaches offer the potential to identify all microbes from all the biological kingdoms. Nucleic acid based pathogen diagnostics has evolved significantly over the past decades. Novel technologies offer increased potential in sensitivity, specificity, decreased costs and parallel sample management. However, most methods are confined to core laboratory facilities. To construct an ultimate nucleic acid based diagnostic for use in areas of need, potential frontline techniques need to be identified and combined. The research focus of this doctoral thesis work has been to develop and apply nucleic acid based methods for pathogen diagnostics. Methods and assays were applied to the two distinct systems i) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae, and ii) genotype determination of the cancer causative Human Papillomavirus (HPV). The first part of the study included development of rapid, direct and multiplex Pyrosequencing nucleic acid screenings. With improved methodology in the sample preparation process, we could detect an existence of multiple co-infecting HPV genotypes at greater sensitivities than previously described, when using the same type of methodology. The second part of the study focused on multiplex nucleic acid amplification strategies using Molecular Inversion Probes with end-step Pyrosequencing screening. The PathogenMip assay presents a complete detection schematic for virtually any known pathogenic organism. We also introduce the novel Connector Inversion Probe, a padlock probe capable of complete gap-fill reactions for multiplex nucleic acid amplifications.
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| 5. |
- Akhras, Michael S., et al.
(författare)
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The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:10
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Tidskriftsartikel (refereegranskat)abstract
- Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e. g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.
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| 6. |
- Gharizadeh, Baback, et al.
(författare)
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Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing
- 2005
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 26:6, s. 486-490
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Tidskriftsartikel (refereegranskat)abstract
- Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.
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| 7. |
- Gharizadeh, Baback, et al.
(författare)
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Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses
- 2006
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Ingår i: Mol Cell Probes. - : Elsevier BV. ; 20:3-4, s. 230-238
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Tidskriftsartikel (refereegranskat)abstract
- Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific 'sentinel' base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.
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| 8. |
- Lindbäck, Emma, et al.
(författare)
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Pyrosequencing of the DNA gyrase gene in Neisseria species : effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae
- 2006
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 114:12, s. 837-841
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Tidskriftsartikel (refereegranskat)abstract
- The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.
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| 9. |
- Pourmand, Nader
(författare)
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RNA-protein complexes as autoantigens : cell and molecular biology of Ro/SSA and disease associations of anti-Ro/SSA antibodies
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Autoantibodies to the Ro/SSA and La/SSB antigens are present in sera from patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The Ro/SSA autoantigen consists of a 52kD and a 60kD protein, complexed with one of four small RNAs. The La protein can also associate with the complex. We have focused mainly on characterisation of Ro 52kD, partly on identification of additional components of the Ro/SSA complex, and partly on the characterisation of anti-Ro and anti-La antibody responses. To study the intracellular localisation of Ro 52kD, expression plasmids were constructed encoding fusion proteins between Ro 52kD subfragments and green fluorescent protein (GFP) from jelly fish. These experiments demonstrated a cytoplasmic localisation of the Ro 52kD protein and indicated a crucial role for the hydrophilic domain in restricting the Ro 52kD protein to the cytoplasm. In order to investigate if the putative zinc fingers of Ro 52kD bind Zn2+ various Ro 52kD subclones were expressed as recombinant proteins and assayed for Zn2+ binding in vitro. One fragment containing the first cysteine/histidine cluster at residues 14-54 and other larger overlapping fragments were demonstrated to bind Zn2+. We also demonstrated that the Zn2+ binding domain is a target for conformation-dependent anti-Ro 52kD autoantibodies. Interestingly, antigenicity is dramatically increased by the reducing conditions which also promote Zn2+ binding. With the aim of searching for additional components of the Ro/SSA complex, a phage display using Ro 60kD and Ro 52kD recombinant fusion proteins as bait proteins was performed on a randomly cloned cDNA library. One candidate gene, phox 40kD, was selected for investigation of the protein-protein interaction with recombinant Ro and La proteins. Further analysis is needed for confirmation of this interaction. Autoantibodies with Ro 52kD, Ro 60kD and La specificities, respectively, were defined within ANA-negative sera. Samples from ANA-negative but Ro/SSA positive patients undergoing investigation due to suspected connective tissue diseases were analysed by immunoblotting and ELISA using recombinant antigens and synthetic peptides. Autoantibodies to Ro 52kD were present in 65% of selected sera and an autoepitope within the Ro 52kD protein composed of the leucine zipper domain was defined. IgA autoantibodies to the Ro 52kD, Ro 60kD and La antigens in sera from patients with primary SS and SLE were analysed using a semiquantitative immunoblotting approach. Our data suggest that IgG dominates the autoantibody response followed by IgM > IgA, and 50-80% of the patients with primary SS and SLE had IgA autoantibodies to the three Ro/SSA proteins. The epitope specificity of the IgA autoantibodies was similar to that of IgG and IgM, and the immunodominant epitopes were represented by aa 136-227 for Ro 52 and by aa 181-320 for Ro 60. In conclusion. the subcellular localisation of Ro 52kD was defined as cytoplasmic and the putative zinc-binding capacity of Ro 52kD demonstrated. IgA autoantibodies to Ro/La were shown to be present in most of Ro/SSA positive sera, and selected ANA-negative sera demonstrated to contain Ro antibodies.
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| 10. |
- Sandberg, Julia
(författare)
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Massively parallel analysis of cells and nucleic acids
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 11. |
- Unemo, Magnus, et al.
(författare)
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Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain
- 2007
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Ingår i: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS). - : Wiley. - 0903-4641 .- 1600-0463. ; 115:3, s. 231-240
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Tidskriftsartikel (refereegranskat)abstract
- A highly discriminative and objective genetic characterization of N. gonorrhoeae, which increases our knowledge of strain populations in different geographic areas, is crucial for the development of improved control measures. In the present study, conventional phenotypic characterization and genetic characterization by means of pulsed-field gel electrophoresis (PFGE), sequencing of the entire porB gene, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and pyrosequencing of a quinolone resistance determining region (QRDR) of the gyrA gene of Swedish ciprofloxacin-resistant N. gonorrhoeae serovar IB-10 isolates (n=45) were performed. The genetic characterization identified one widely spread ciprofloxacin-resistant N. gonorrhoeae ST147 strain. In addition, isolates with slightly different genetic characteristics, which presumably reflect the ongoing evolution only, were also identified. All the isolates contained single nucleotide polymorphisms in QRDR of the gyrA gene that are highly correlated with ciprofloxacin resistance. Consequently, comprehensive characterization identified the first confirmed large domestic transmission, mainly among young heterosexuals, of one ciprofloxacin-resistant N. gonorrhoeae strain in Swedish society during 2002-2003. In conclusion, a precise, i. e. genetic, characterization for identification of individual strains is a very valuable support to the crucial active surveillance of the epidemiological characteristics and the antibiotic susceptibility of N. gonorrhoeae in the effective treatment of gonorrhoea.
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