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Targeted resequencing of candidate genes using Selector Probes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. (Forskargrupp Mats Nilsson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 2, e8- p.Article in journal (Refereed) Published
Abstract [en]

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94 specificity and 98 coverage of the targeted region was achieved. Reproducibility between replicates was high (R 2=0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.

Place, publisher, year, edition, pages
2011. Vol. 39, no 2, e8- p.
Keyword [en]
Resequencing, Targeted, Next generation sequencing, Selectors, Cancer, Tumor, FFPE
National Category
Medical and Health Sciences
Research subject
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-121425DOI: 10.1093/nar/gkq1005ISI: 000286675300003PubMedID: 21059679OAI: oai:DiVA.org:uu-121425DiVA: diva2:305278
Available from: 2010-03-23 Created: 2010-03-23 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Extracting Genomic Variations using Selector Technology
Open this publication in new window or tab >>Extracting Genomic Variations using Selector Technology
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis describes the development and use of a new class of molecular tools called Selector probes, and its potential for investigations of genetic variation. The Selector technology provides multiplex amplification of targeted DNA sequences with a high specificity, and an enrichment factor in the same order of magnitude as PCR. A common feature in this thesis work is to focus the analysis on DNA regions of interest. For example, this technique can be implemented in analysing candidate regions found by whole genome studies that need validation (global to local analysis), and applications requiring detection of rare alleles (common to rare allele), important in for example cancer samples.

An assay is presented that allows for fast and simple quantification of relative copy-number variations. The method was proven to be able to detect aneuploidy in chromosome 13, 18, 21 and X, with a resolution enough to distinguish between 4 and 5 copies. The method was successfully applied to solve a biological question regarding a copy-number variation, that explains the Ridge phenotype typical for the dog bread Rhodesian Ridgebacks. The Selector strategy was able to detect and map a tandem duplication with a size of 133 kb, which was characterized with base-pair resolution.

A readout platform that facilitates simultaneous digital quantitative analysis of a large numbers of biomolecules is further introduced. The work involves arraying amplified product from successful selection and decoding each molecule by hybridization of fluorophore labeled oligonucleotides.

Finally, a genome partitioning method which is applied upstream of next generation sequencing platforms is presented. It is shown that the method provides successful enrichment with 98 % coverage and 94 % specificity and high enrichment uniformity. The technique was applied for mutation analysis of 26 cancer-related genes in tumor cell-lines and tissue.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 46 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 543
Keyword
Selector, Selector probe, Genetic variation, Resequencing, Targeted sequencing, copy-number variations, MLGA, Bioinformatics, Next generation sequencing, NGS, Amplified single molecule, ASM
National Category
Medical Genetics
Research subject
Genetics; Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-121429 (URN)978-91-554-7761-5 (ISBN)
Public defence
2010-05-07, Rudbeckhall, Rudbeck Laboratory, Dag Hammarskjölds väg 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-14 Created: 2010-03-23 Last updated: 2018-01-12
2. Microfluidic and Molecular Tools for Genetic Analyses
Open this publication in new window or tab >>Microfluidic and Molecular Tools for Genetic Analyses
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Methods that enable interrogation of multiple genomic regions in parallel are very useful for efficient detection of genetic variation. Two different types of probes are described in this thesis that can be used for direct analysis or for sample preparation upstream of Next Generation Sequencing.  In addition to the development of molecular probing systems it also reports on the progress of two assay formats for biological experiments.

The Selector probe enrich for genomic regions of interest by probe mediated specific circularization of target fragments. Amplification based enrichment of circles can be carried out using polymerase chain reaction, rolling-circle amplification or multiple displacement amplification. Enrichment of all exons in 28 genes known to be mutated in lung and/or colon cancer is demonstrated.  Selection and analysis by SOLiD Sequencing was performed on fresh frozen and formalin fixed paraffin embedded (FFPE) samples, and mutations previously detected by Sanger sequencing were detected.  The extractor probe is another probe variant that can be used for multiplex enrichment of DNA. It targets genomic fragments by using both ligation and sequence specific elongation for discrimination between on and off target sequences.

A microfluidic platform fabricated by compact disc injection molding that can be used for biological assays is described.  Microchannel structures in thermoplastic material are coated with silicon dioxide by electron beam evaporation which facilitates closing of the structures by PDMS- glass bonding by ozone plasma. The platform’s utility for biological experiments is demonstrated by for detection of amplified single molecules (ASM), cell culturing and on-chip peristaltic pumping.

The thesis also includes an exploratory study for the purpose of using a non-optical system for detection of ASM’s.  Optimizations were performed of the conditions needed in order to detect an increase in hydrodynamic size of magnetic particles, using a superconducting quantum interference device (SQUID), as they form complex with ASM’s.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 36 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 546
Keyword
microfluidics, PCR, sequencing, cancer genetics
National Category
Medical Genetics
Research subject
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-121536 (URN)978-91-554-7765-3 (ISBN)
Public defence
2010-05-05, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2010-04-14 Created: 2010-03-24 Last updated: 2018-01-12Bibliographically approved

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Isaksson, MagnusGöransson Kultima, Hanna

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