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Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 12, e52750- p.Article in journal (Refereed) Published
Abstract [en]

The genomic revolution in oncology will entail mutational analyses of vast numbers of patient-matched tumor and normal tissue samples. This has meant an increased risk of patient sample mix up due to manual handling. Therefore, scalable genotyping and sample identification procedures are essential to pathology biobanks. We have developed an efficient alternative to traditional genotyping methods suited for automated analysis. By targeting 53 prevalent deletions and insertions found in human populations with fluorescent multiplex ligation dependent genome amplification, followed by separation in a capillary sequencer, a peak spectrum is obtained that can be automatically analyzed. 24 tumor-normal patient samples were successfully matched using this method. The potential use of the developed assay for forensic applications is discussed.

Place, publisher, year, edition, pages
2012. Vol. 7, no 12, e52750- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-193620DOI: 10.1371/journal.pone.0052750ISI: 000312829100077OAI: oai:DiVA.org:uu-193620DiVA: diva2:603459
Available from: 2013-02-06 Created: 2013-02-05 Last updated: 2017-12-06Bibliographically approved
In thesis
1. From Tissue to Mutations: Genetic Profiling of Colorectal Cancer
Open this publication in new window or tab >>From Tissue to Mutations: Genetic Profiling of Colorectal Cancer
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Comprehensive characterisation of the mutational landscapes of solid tumours is a multistep process involving the collection of suitable samples, the extraction of nucleic acids and the preparation of these materials for mutational analyses. In this thesis, I aimed to develop a streamlined process for the analysis of colorectal cancer (CRC) patient samples in order to identify novel mutations that hallmark the development of advanced disease.

Papers I and II outline a technique for serial extraction of nucleic acids from frozen tissue that we developed and subsequently implemented on a robotic platform to enable high-throughput processing. The extracted nucleic acids were validated in downstream processes relevant for genetic analyses, including traditional Sanger and next generation sequencing  techniques.

In Paper III, we developed a genotyping method based on multiplex ligation-dependent genome amplification. The method was designed such that InDel polymorphisms of between 30 and 70 % prevalence in a European population were selected and amplified in a multiplex PCR assay. DNA from 24 patient-matched colorectal tumour and normal tissues was genotyped and paired with a high match probability.

In Paper IV, we performed targeted resequencing of 107 primary CRCs, of which approximately half developed metastatic disease or had distant metastases at the time of diagnosis. We chose to analyse 676 genes based on their involvement in key signalling pathways in CRC. We found an enrichment of mutations in the Eph receptor tyrosine kinase gene family in metastatic patients, indicating a potential role for these genes in CRC metastasis.

This thesis outlines a series of procedures that can be employed in a high-throughput setting for the analysis of solid tumours. We applied these methods to the analysis of colorectal tumours and propose a link between novel somatic mutations and metastatic disease.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1028
Keyword
Nucleic acid extraction, genotyping, targeted sequencing, mutation analysis, colorectal cancer, metastatic disease
National Category
Medical Genetics Cancer and Oncology
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-231508 (URN)978-91-554-9042-3 (ISBN)
Public defence
2014-11-07, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
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Supervisors
Available from: 2014-10-16 Created: 2014-09-08 Last updated: 2015-01-23

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Mathot, LucyFalk Sörqvist, ElinMoens, LotteAllen, MarieSjöblom, TobiasNilsson, Mats

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