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Assessment of Whole Genome Amplification for Sequence Capture and Massively Parallel Sequencing
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, e84785- p.Article in journal (Refereed) Published
Abstract [en]

Exome sequence capture and massively parallel sequencing can be combined to achieve inexpensive and rapid global analyses of the functional sections of the genome. The difficulties of working with relatively small quantities of genetic material, as may be necessary when sharing tumor biopsies between collaborators for instance, can be overcome using whole genome amplification. However, the potential drawbacks of using a whole genome amplification technology based on random primers in combination with sequence capture followed by massively parallel sequencing have not yet been examined in detail, especially in the context of mutation discovery in tumor material. In this work, we compare mutations detected in sequence data for unamplified DNA, whole genome amplified DNA, and RNA originating from the same tumor tissue samples from 16 patients diagnosed with non-small cell lung cancer. The results obtained provide a comprehensive overview of the merits of these techniques for mutation analysis. We evaluated the identified genetic variants, and found that most (74%) of them were observed in both the amplified and the unamplified sequence data. Eighty-nine percent of the variations found by WGA were shared with unamplified DNA. We demonstrate a strategy for avoiding allelic bias by including RNA-sequencing information.

Place, publisher, year, edition, pages
2014. Vol. 9, no 1, e84785- p.
Keyword [en]
Exome Capture, Amplified Dna, Mutation, Cancer, Metagenomes, Discovery, Viruses, Samples, Tumor
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-104473DOI: 10.1371/journal.pone.0084785ISI: 000329463500033Scopus ID: 2-s2.0-84896910544OAI: oai:DiVA.org:kth-104473DiVA: diva2:564857
Funder
EU, European Research Council, CHEMORES LSHC-CT-2007-037665Swedish Cancer SocietySwedish Research CouncilScience for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140211. Updated from submitted to published.

Available from: 2012-11-05 Created: 2012-11-05 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Analysis of genetic variations in cancer
Open this publication in new window or tab >>Analysis of genetic variations in cancer
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this thesis is to apply recently developed technologies for genomic variation analyses, and to ensure quality of the generated information for use in preclinical cancer research.

Faster access to a patients’ full genomic sequence for a lower cost makes it possible for end users such as clinicians and physicians to gain a more complete understanding of the disease status of a patient and adjust treatment accordingly. Correct biological interpretation is important in this context, and can only be provided through fast and simple access to relevant high quality data.

Therefore, we here propose and validate new bioinformatic strategies for biomarker selection for prediction of response to cancer therapy. We initially explored the use of bioinformatic tools to select interesting targets for toxicity in carboplatin and paclitaxel on a smaller scale. From our findings we then further extended the analysis to the entire exome to look for biomarkers as targets for adverse effects from carboplatin and gemcitabine. To investigate any bias introduced by the methods used for targeting the exome, we analyzed the mutation profiles in cancer patients by comparing whole genome amplified DNA to unamplified DNA. In addition, we applied RNA-seq to the same patients to further validate the variations obtained by sequencing of DNA. The understanding of the human cancer genome is growing rapidly, thanks to methodological development of analysis tools. The next step is to implement these tools as a part of a chain from diagnosis of patients to genomic research to personalized treatment.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. iii, 62 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2012:18
Keyword
Cancer, Mutations, Variations, Single Nucleotide Polymorphism, DNA, RNA, Genome, Massively Parallel Sequencing, Exome Sequencing, Toxicity
National Category
Other Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-104438 (URN)978-91-7501-450-0 (ISBN)
Public defence
2012-11-22, Hillarpsalen, Retzius väg 8, Karolinska Institutet, Solna, 09:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121105

Available from: 2012-11-05 Created: 2012-11-02 Last updated: 2014-02-11Bibliographically approved

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