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Increased Throughput by Parallelization of Library Preparation for Massive Sequencing
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
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2010 (English)In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 3, e10029- p.Article in journal (Refereed) Published
Abstract [en]

Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.

Place, publisher, year, edition, pages
2010. Vol. 5, no 3, e10029- p.
Keyword [en]
POLYETHYLENE-GLYCOL, DNA, PRECIPITATION
National Category
Other Industrial Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-28306DOI: 10.1371/journal.pone.0010029ISI: 000276420400007Scopus ID: 2-s2.0-77956313182OAI: oai:DiVA.org:kth-28306DiVA: diva2:386726
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Note
QC 20110113Available from: 2011-01-13 Created: 2011-01-12 Last updated: 2012-11-16Bibliographically approved
In thesis
1. Enabling massive genomic and transcriptomic analysis
Open this publication in new window or tab >>Enabling massive genomic and transcriptomic analysis
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In recent years there have been tremendous advances in our ability to rapidly and cost-effectively sequence DNA. This has revolutionized the fields of genetics and biology, leading to a deeper understanding of the molecular events in life processes. The rapid advances have enormously expanded sequencing opportunities and applications, but also imposed heavy strains on steps prior to sequencing, as well as the subsequent handling and analysis of the massive amounts of sequence data that are generated, in order to exploit the full capacity of these novel platforms. The work presented in this thesis (based on six appended papers) has contributed to balancing the sequencing process by developing techniques to accelerate the rate-limiting steps prior to sequencing, facilitating sequence data analysis and applying the novel techniques to address biological questions.

 

Papers I and II describe techniques to eliminate expensive and time-consuming preparatory steps through automating library preparation procedures prior to sequencing. The automated procedures were benchmarked against standard manual procedures and were found to substantially increase throughput while maintaining high reproducibility. In Paper III, a novel algorithm for fast classification of sequences in complex datasets is described. The algorithm was first optimized and validated using a synthetic metagenome dataset and then shown to enable faster analysis of an experimental metagenome dataset than conventional long-read aligners, with similar accuracy. Paper IV, presents an investigation of the molecular effects on the p53 gene of exposing human skin to sunlight during the course of a summer holiday. There was evidence of previously accumulated persistent p53 mutations in 14% of all epidermal cells. Most of these mutations are likely to be passenger events, as the affected cell compartments showed no apparent growth advantage. An annual rate of 35,000 novel sun-induced persistent p53 mutations was estimated to occur in sun-exposed skin of a human individual.  Paper V, assesses the effect of using RNA obtained from whole cell extracts (total RNA) or cytoplasmic RNA on quantifying transcripts detected in subsequent analysis. Overall, more differentially detected genes were identified when using the cytoplasmic RNA. The major reason for this is related to the reduced complexity of cytoplasmic RNA, but also apparently due (at least partly) to the nuclear retention of transcripts with long, structured 5’- and 3’-untranslated regions or long protein coding sequences. The last paper, VI, describes whole-genome sequencing of a large, consanguineous family with a history of Leber hereditary optic neuropathy (LHON) on the maternal side. The analysis identified new candidate genes, which could be important in the aetiology of LHON. However, these candidates require further validation before any firm conclusions can be drawn regarding their contribution to the manifestation of LHON.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. 45 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:24
Keyword
DNA, RNA, sequencing, massively parallel sequencing, alignment, assembly, single nucleotide polymorphism, LHON
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-45957 (URN)978-91-7501-164-6 (ISBN)
Public defence
2011-12-02, Petrén‐salen, Nobels väg 12B, Karolinska Institute Campus Solna, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20111115Available from: 2011-11-15 Created: 2011-11-01 Last updated: 2011-11-15Bibliographically approved
2. 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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