Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Methods to Prepare DNA for Efficient Massive Sequencing
KTH, Skolan för bioteknologi (BIO), Genteknologi.
2012 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Stockholm: KTH Royal Institute of Technology, 2012. , s. ii, 61
Serie
Trita-BIO-Report, ISSN 1654-2312 ; 2012:22
Emneord [en]
DNA, Massive sequencing, Next Generation Sequencing, Library Preparation, Barcoding, Multiplexing
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-105116ISBN: 978-91-7501-548-4 (tryckt)OAI: oai:DiVA.org:kth-105116DiVA, id: diva2:570053
Disputas
2012-12-07, Gardaulan, Smittshyddsinstitutet, Nobels väg 18, Solna, 10:00 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20121126

Tilgjengelig fra: 2012-11-16 Laget: 2012-11-16 Sist oppdatert: 2022-06-24bibliografisk kontrollert
Delarbeid
1. Increased Throughput by Parallelization of Library Preparation for Massive Sequencing
Åpne denne publikasjonen i ny fane eller vindu >>Increased Throughput by Parallelization of Library Preparation for Massive Sequencing
Vise andre…
2010 (engelsk)Inngår i: PLOS ONE, ISSN 1932-6203, Vol. 5, nr 3, s. e10029-Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
POLYETHYLENE-GLYCOL, DNA, PRECIPITATION
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-28306 (URN)10.1371/journal.pone.0010029 (DOI)000276420400007 ()20386591 (PubMedID)2-s2.0-77956313182 (Scopus ID)
Forskningsfinansiär
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Merknad
QC 20110113Tilgjengelig fra: 2011-01-13 Laget: 2011-01-12 Sist oppdatert: 2024-03-18bibliografisk kontrollert
2. Large Scale Library Generation for High Throughput Sequencing Authors and Affiliations
Åpne denne publikasjonen i ny fane eller vindu >>Large Scale Library Generation for High Throughput Sequencing Authors and Affiliations
2011 (engelsk)Inngår i: PLOS ONE, E-ISSN 1932-6203, Vol. 6, nr 4, s. e19119-Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
Cell lines
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-33950 (URN)10.1371/journal.pone.0019119 (DOI)000290019400031 ()21589638 (PubMedID)2-s2.0-79955691833 (Scopus ID)
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad
QC 20110609Tilgjengelig fra: 2011-06-09 Laget: 2011-05-23 Sist oppdatert: 2024-03-18bibliografisk kontrollert
3. Decoding a substantial set of samples in parallel by massive sequencing
Åpne denne publikasjonen i ny fane eller vindu >>Decoding a substantial set of samples in parallel by massive sequencing
2011 (engelsk)Inngår i: Plos One, ISSN 1932-6203, Vol. 6, nr 3Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The dramatic increase of throughput seen in the eld of sequenceanalysis during the last years has opened up new possibilities of se-quencing a multitude of samples in parallel. Here we present a novelstrategy where the combination of two tags is used to link reads totheir origins in a pool of samples. The two tags are incorporated intwo steps leading to lowering of sample handling complexity by nearly100 times. The method described here enables accurate identi cationand typing of thousands of samples in parallel and is scalable. In thisstudy the system was designed to test 4992 samples using only 122 tags.

To proof the concept of two tagging method the highly polymor-phic 2nd exon of DLA-DRB1 in dogs and wolves was sequenced usingthe 454 GS FLX Titanium Chemistry. By requiring a minimum se-quence depth of 20 reads per sample, 94% of the successfully ampli edsamples were genotyped. In addition, the method allowed digital de-tection of chimeric fragments. These results demonstrate that it ispossible to sequence thousands of samples in parallel without complexpooling patterns or primer combinations. Furthermore, the method isscalable and increasing the sample size by 960 samples requires only10 additional tags.

sted, utgiver, år, opplag, sider
San Fransisco: PUBLIC LIBRARY SCIENCE, 2011
Emneord
HIGH-THROUGHPUT; HIGH-RESOLUTION; DNA; PROBES
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-24353 (URN)10.1371/journal.pone.0017785 (DOI)000288170900047 ()21408018 (PubMedID)2-s2.0-79952430921 (Scopus ID)
Forskningsfinansiär
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Merknad

QC 20100906 Uppdaterad från manuskript till artikel i tidskrift 20110407

Tilgjengelig fra: 2010-09-06 Laget: 2010-09-03 Sist oppdatert: 2024-03-18bibliografisk kontrollert
4. Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
Åpne denne publikasjonen i ny fane eller vindu >>Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
Vise andre…
2013 (engelsk)Inngår i: Scientific Reports, E-ISSN 2045-2322, Vol. 3, s. 1186-Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
Human Genome, Structural Variation, Domestic Dog, Dna, Mutations
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-105124 (URN)10.1038/srep01186 (DOI)000315767100001 ()23470464 (PubMedID)2-s2.0-84875360897 (Scopus ID)
Forskningsfinansiär
EU, FP7, Seventh Framework Programme, 222913Swedish Research Council
Merknad

QC 20130405. Updated from submitted to published.

Tilgjengelig fra: 2012-11-16 Laget: 2012-11-16 Sist oppdatert: 2024-03-15bibliografisk kontrollert
5. Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
Åpne denne publikasjonen i ny fane eller vindu >>Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
Vise andre…
2015 (engelsk)Inngår i: PLOS ONE, E-ISSN 1932-6203, Vol. 10, nr 1, artikkel-id e0117059Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
Public Library of Science, 2015
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-105132 (URN)10.1371/journal.pone.0117059 (DOI)000348732100060 ()25629514 (PubMedID)2-s2.0-84922424353 (Scopus ID)
Forskningsfinansiär
EU, FP7, Seventh Framework Programme, 222913Swedish Foundation for Strategic Research
Merknad

Updated from Submitted to Published. QC 20150407

Tilgjengelig fra: 2012-11-16 Laget: 2012-11-16 Sist oppdatert: 2024-03-15bibliografisk kontrollert

Open Access i DiVA

fulltext(1584 kB)2515 nedlastinger
Filinformasjon
Fil FULLTEXT01.pdfFilstørrelse 1584 kBChecksum SHA-512
fda9f0a69caf97e683b858453cda07ec22101b3dfc83673bfa35fcf7c4be2761220e1d163c8e3d22f7cf62e9736b521d35776db86d0325ae08ec1c3a77ce2fa0
Type fulltextMimetype application/pdf

Søk i DiVA

Av forfatter/redaktør
Lundin, Sverker
Av organisasjonen

Søk utenfor DiVA

GoogleGoogle Scholar
Totalt: 2693 nedlastinger
Antall nedlastinger er summen av alle nedlastinger av alle fulltekster. Det kan for eksempel være tidligere versjoner som er ikke lenger tilgjengelige

isbn
urn-nbn

Altmetric

isbn
urn-nbn
Totalt: 633 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf