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Methods for Analyzing Genomes
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0002-2207-7370
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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

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

Place, publisher, year, edition, pages
2010. , 53 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2010:4
Keyword [en]
array, sequence capture, genotyping, trinucleotide threading, sequencing, massively parallel sequencing, single molecule sequencing, Visual DNA, p53, single nucleotide polymorphism, biomarker
National Category
Genetics
Identifiers
URN: urn:nbn:se:kth:diva-12407ISBN: 978-91-7415-596-9 (print)OAI: oai:DiVA.org:kth-12407DiVA: diva2:310884
Public defence
2010-05-07, FD5, AlbaNova Universitetscentrum, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2010-04-19 Created: 2010-04-16 Last updated: 2010-08-26
List of papers
1. Visual DNA: Identification of DNA sequence variations by bead trapping
Open this publication in new window or tab >>Visual DNA: Identification of DNA sequence variations by bead trapping
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2007 (English)In: Genomics, ISSN 0888-7543, E-ISSN 1089-8646, Vol. 90, 741-745 p.Article in journal (Refereed) Published
Abstract [en]

In this paper we describe a method that uses the nearly covalent strength biotin-streptavidin interaction to attach a paramagnetic bead of micrometer size to a DNA molecule of nanometer size, scaling up the spatial size of a query DNA strand by a factor of 1000, making it visible to the human eye. The use of magnetic principles enables rapid binding and washing of detector beads, facilitating a readout of amplified DNA sequences in a few minutes. Here we exemplify the method on mitochondrial DNA variations using an array platform. Visual identification and documentation can be performed with ail ordinary mobile phone equipped with a built-in camera.

Keyword
imaging techniques; medical imaging; magnetics; microspheres; oligonucleotide array; sequence analysis; signal amplification assay
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-11218 (URN)10.1016/j.ygeno.2007.07.014 (DOI)000251707500010 ()
Note
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Visual DNA as a diagnostic tool
Open this publication in new window or tab >>Visual DNA as a diagnostic tool
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2009 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 30, no 21, 3691-3695 p.Article in journal (Refereed) Published
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.

Keyword
Cystic fibrosis, Diagnostics, Mutation, PrAse, Visual DNA, CYSTIC-FIBROSIS GENE, IDENTIFICATION, TECHNOLOGIES
Identifiers
urn:nbn:se:kth:diva-12401 (URN)10.1002/elps.200900273 (DOI)000272126000006 ()2-s2.0-70649091306 (Scopus ID)
Note
qc 20100416Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2017-12-12Bibliographically approved
3. Flow cytometry for enrichment and titration in massively parallel DNA sequencing
Open this publication in new window or tab >>Flow cytometry for enrichment and titration in massively parallel DNA sequencing
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2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 8Article in journal (Refereed) Published
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.

Identifiers
urn:nbn:se:kth:diva-12402 (URN)10.1093/nar/gkp188 (DOI)000265953000007 ()2-s2.0-65849474143 (Scopus ID)
Note
QC 20100416Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2017-12-12Bibliographically approved
4. Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads
Open this publication in new window or tab >>Allelotyping by Massively Parallel Pyrosequencing of SNP-carrying Trinucleotide Threads
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2008 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 29, no 2, 323-329 p.Article in journal (Refereed) Published
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.

Keyword
trinucleotide threading; pooled genomic DNA; association studies; multiplexing; SNP; allelotyping; cancer; obesity
National Category
Other Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-7690 (URN)10.1002/humu.20655 (DOI)000253033000016 ()2-s2.0-38949145121 (Scopus ID)
Note
QC 20100416Available from: 2007-11-21 Created: 2007-11-21 Last updated: 2017-12-14Bibliographically approved
5. Sun‐induced Missense Mutations Are Extensively Accumulated and Tolerated in Phenotypically Intact Stem Cell Compartments of Human Skin
Open this publication in new window or tab >>Sun‐induced Missense Mutations Are Extensively Accumulated and Tolerated in Phenotypically Intact Stem Cell Compartments of Human Skin
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(English)Article in journal (Refereed) Submitted
Abstract [en]

Here we demonstrate that intermittently sun‐exposed human skin contains an extensive number of phenotypically intact stem cell compartments bearing missense mutations in the p53 tumor suppressor gene. Deep sequencing of sun‐exposed and shielded microdissected skin from mid‐life individuals revealed that persistent p53 mutations had accumulated in 14% of all epidermal cells, with no apparent signs of a growth advantage of the affected cell compartments. Furthermore, 6% of the mutated epidermal cells encoded a truncated protein. The abundance of these events, not taking into account intron mutations and mutations in other genes that also may have functional implications, suggests an extensive tolerance of human cells to severe genetic alterations caused by ultraviolet light, with an estimated annual rate of accumulation of approximately 35,000 new persistent protein altering p53 mutations in sun exposed skin of a human individual.

Identifiers
urn:nbn:se:kth:diva-12404 (URN)
Note
QC 20100416Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2011-11-15Bibliographically approved
6. Discovery of breast cancer biomarker candidates by translational database selection and multiplex enrichment strategies
Open this publication in new window or tab >>Discovery of breast cancer biomarker candidates by translational database selection and multiplex enrichment strategies
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(English)Manuscript (preprint) (Other academic)
Identifiers
urn:nbn:se:kth:diva-12406 (URN)
Note
QC 20100416Available from: 2010-04-16 Created: 2010-04-16 Last updated: 2010-06-18Bibliographically approved

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