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Hierarchical molecular tagging to resolve long continuous sequences by 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.
Science for Life Laboratory, Stockholm University, Department of Biochemistry and Biophysics, Stockholm.
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2013 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, 1186- p.Article in journal (Refereed) Published
Abstract [en]

Here we demonstrate the use of short-read massive sequencing systems to in effect achieve longer read lengths through hierarchical molecular tagging. We show how indexed and PCR-amplified targeted libraries are degraded, sub-sampled and arrested at timed intervals to achieve pools of differing average length, each of which is indexed with a new tag. By this process, indices of sample origin, molecular origin, and degree of degradation is incorporated in order to achieve a nested hierarchical structure, later to be utilized in the data processing to order the reads over a longer distance than the sequencing system originally allows. With this protocol we show how continuous regions beyond 3000 bp can be decoded by an Illumina sequencing system, and we illustrate the potential applications by calling variants of the lambda genome, analysing TP53 in cancer cell lines, and targeting a variable canine mitochondrial region.

Place, publisher, year, edition, pages
2013. Vol. 3, 1186- p.
Keyword [en]
Human Genome, Structural Variation, Domestic Dog, Dna, Mutations
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-105124DOI: 10.1038/srep01186ISI: 000315767100001Scopus ID: 2-s2.0-84875360897OAI: oai:DiVA.org:kth-105124DiVA: diva2:570077
Funder
EU, FP7, Seventh Framework Programme, 222913Swedish Research Council
Note

QC 20130405. Updated from submitted to published.

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

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