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Chemical fragmentation for massively parallel sequencing library preparation
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.
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.
2013 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 168, no 1, 95-100 p.Article in journal (Refereed) Published
Abstract [en]

Fragmentation is essential in most library preparation protocols for use with massively parallel sequencing systems. Complexes that generate hydroxyl radicals, such as iron-EDTA, can be used to introduce random DNA cleavage. Here we describe a chemical fragmentation method that can be incorporated into library preparation protocols for next-generation sequencing workflows. This protocol has been validated by whole genome, amplicon and exome sequencing. Chemical fragmentation is a cost-effective alternative to current fragmentation methods that has no observable sequence bias and requires no instrumentation.

Place, publisher, year, edition, pages
2013. Vol. 168, no 1, 95-100 p.
Keyword [en]
Fragmentation, High-throughput sequencing, Library preparation
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-133269DOI: 10.1016/j.jbiotec.2013.08.020ISI: 000325464300016Scopus ID: 2-s2.0-84883827621OAI: oai:DiVA.org:kth-133269DiVA: diva2:660485
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscienceSwedish Research CouncilSwedish Foundation for Strategic Research
Note

QC 20131030

Available from: 2013-10-30 Created: 2013-10-29 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Advances in DNA Detection
Open this publication in new window or tab >>Advances in DNA Detection
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

DNA detection technologies have an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis, food safety evaluation, and environmental monitoring. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used in research laboratories, in clinical and forensic practice.

The first aim of this thesis is to unravel the potential of DNA detection on cellulose filter paper and further investigate the filter paper as a viable candidate for DNA array support. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on the active paper using fluorescence. In Paper II, we addressed visual detection with magnetic beads and increased the detection throughput on the active filter paper, which required no instrumentation. Second, in pursuit of a rapid, sensitive and specific pathogen diagnosis in bloodstream infection (BSI), we explored the possibility of rare DNA detection in the presence of a high amount of background DNA by an enzymatic reaction, which can remove background DNA while enriching the rare DNA fraction. In order to overcome the challenge of the second objective, we developed a chemical fragmentation method to increase the efficiency of enzymatic digestion and hybridization. In addition, DNA library preparation for massively parallel sequencing may benefit from the chemical fragmentation. Paper III and Paper IV introduce this work.

The findings in Paper I showed that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups, while preserving the base pairing ability of the oligonucleotides. In Paper II, visual detection of DNA on active paper was achieved without instrumentation, based on the natural colour of magnetic beads. Furthermore, the possibility to increase the throughput of DNA detection on active paper was demonstrated by successful multiplex detection. In Paper III, the developed chemical fragmentation was verified to be suitable for DNA library preparation in massively parallel sequencing. The fragmentation technique is simple to perform, cost-effective and amenable to automation. In Paper IV, a limited amount of E.coli DNA was detected amid a much larger amount of human background DNA in a BSI model, which comprises of human and E.coli amplicons with an abundance ratio of 108. Human β-actin amplicons were suppressed 105-fold, whereas the E.coli amplicons remained unaffected. The model system was applied to and improved with clinical plasma and blood samples from septic patients.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2014. ix, 56 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2014:4
Keyword
DNA detection, active filter paper, visual detection, throughput, fluorescence, superparamagnetic beads, rare DNA, Q-PCR, chemical fragmentation, DNA library preparation, massively parallel sequencing
National Category
Other Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-143047 (URN)978-91-7595-053-2 (ISBN)
Public defence
2014-04-11, CMB Lecture hall, Berzelius väg 21, Karolinska Institute, Solna, 10:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20140318

Available from: 2014-03-18 Created: 2014-03-16 Last updated: 2014-03-18Bibliographically approved

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