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Large Scale Library Generation for High Throughput Sequencing Authors and Affiliations
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.ORCID iD: 0000-0003-4313-1601
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 4, e19119- p.Article in journal (Refereed) Published
Abstract [en]

Background: Large efforts have recently been made to automatethe sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols. Methodology/Principal Findings: In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitation of certain sizes of DNA molecules on to paramagnetic beads for cleanup and selection after standard enzymatic reactions. Conclusions/Significance: The method is used to generate libraries for de novo and re-sequencing on the Illumina HiSeq 2000 instrument with a throughput of 12 samples per instrument in approximately 4 hours. The resulting output data show quality scores and pass filter rates comparable to manually prepared samples. The sample size distribution can be adjusted for each application, and are suitable for all high throughput DNA processing protocols seeking to control size intervals.

Place, publisher, year, edition, pages
2011. Vol. 6, no 4, e19119- p.
Keyword [en]
Cell lines
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-33950DOI: 10.1371/journal.pone.0019119ISI: 000290019400031Scopus ID: 2-s2.0-79955691833OAI: oai:DiVA.org:kth-33950DiVA: diva2:421688
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Note
QC 20110609Available from: 2011-06-09 Created: 2011-05-23 Last updated: 2017-12-11Bibliographically 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. Technologies for Single Cell Genome Analysis
Open this publication in new window or tab >>Technologies for Single Cell Genome Analysis
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules.

As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample.

Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. 48 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2016:1
Keyword
DNA, sequencing, single molecule, single cell, whole genome amplification, exome sequencing, emulsions, barcoding, phasin
National Category
Bioinformatics and Systems Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-181059 (URN)978-91-7595-842-2 (ISBN)
Public defence
2016-02-19, Air and Fire, Science for Life Laboratory, KTH, Tomtebodavägen 23A, Solna, 10:00 (English)
Opponent
Supervisors
Note

QC 20160127

Available from: 2016-01-27 Created: 2016-01-27 Last updated: 2016-01-27Bibliographically approved

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