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Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
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.ORCID iD: 0000-0002-2219-0197
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 1, e0117059Article in journal (Refereed) Published
Abstract [en]

Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.

Place, publisher, year, edition, pages
Public Library of Science , 2015. Vol. 10, no 1, e0117059
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-105132DOI: 10.1371/journal.pone.0117059ISI: 000348732100060Scopus ID: 2-s2.0-84922424353OAI: oai:DiVA.org:kth-105132DiVA: diva2:570085
Funder
EU, FP7, Seventh Framework Programme, 222913Swedish Foundation for Strategic Research
Note

Updated from Submitted to Published. QC 20150407

Available from: 2012-11-16 Created: 2012-11-16 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Methods to Prepare DNA for Efficient Massive Sequencing
Open this publication in new window or tab >>Methods to Prepare DNA for Efficient Massive Sequencing
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity.

The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding.

Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. ii, 61 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2012:22
Keyword
DNA, Massive sequencing, Next Generation Sequencing, Library Preparation, Barcoding, Multiplexing
National Category
Other Industrial Biotechnology Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:kth:diva-105116 (URN)978-91-7501-548-4 (ISBN)
Public defence
2012-12-07, Gardaulan, Smittshyddsinstitutet, Nobels väg 18, Solna, 10:00 (English)
Opponent
Supervisors
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note

QC 20121126

Available from: 2012-11-16 Created: 2012-11-16 Last updated: 2013-04-15Bibliographically approved
2. Library Preparation for High Throughput DNA Sequencing
Open this publication in new window or tab >>Library Preparation for High Throughput DNA Sequencing
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Order 3 billion base pairs of DNA in the correct order and you get the blueprint of a human, the genome. Before the introduction of massively parallel sequencing a little more than a decade ago it would cost around $10 million to get this blueprint. Since then, sequencing throughput and cost have plummeted and now that figure is around $1000, and large sequencing centres such as the National Genomics Infrastructure in Stockholm is sequencing the equivalent of 25 human genomes per hour. The papers that form the basis of this thesis cover different aspects of the rapidly expanding DNA sequencing field.

 

Paper I describes a model system that employ massively parallel sequencing to characterize the behaviour of type IIS restriction enzymes. Enzymes are biological macromolecules that catalyse chemical reactions in the cell. All commercially available sequencing systems use enzymes to prepare the nucleic acids before they are loaded on the machine. Thus, intimate knowledge of enzymes is vital not only when designing new sequencing protocols, but also for understanding the limitations of current protocols. Paper II covers the automation of a library preparation protocol for spatially resolved transcriptome sequencing. Automation increases the sample throughput and also minimises the risk of human errors that can introduce technical noise in the data. In paper III, the power of massively parallel sequencing is employed to describe the RNA content of the endometrium at two different time points during the menstrual cycle. Finally, paper IV covers the sequencing of highly degraded nucleic acids from formalin fixed, paraffin embedded samples. These samples often have a rich clinical background, making them extremely valuable for researchers. However, it is challenging to sequence these samples and this study looks at the impact that different preparation kits have on the quality of the sequencing data. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2017. 56 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2017:1
Keyword
DNA, RNA, sequencing, massively parallel sequencing, library preparation, automation, genome, transcriptome
National Category
Biological Sciences
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-196560 (URN)978-91-7729-212-8 (ISBN)
Public defence
2017-01-13, Air & Fire, Science for Life Laboratory, Tomtebodavägen 23, Solna, 13:00 (English)
Opponent
Supervisors
Note

QC 20161124

Available from: 2016-11-24 Created: 2016-11-16 Last updated: 2016-11-24Bibliographically approved

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