| 1. |
- Lundin, Sverker, et al.
(författare)
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Endonuclease specificity and sequence dependence of Type IIS restriction enzymes
- 2015
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Lundin, Sverker, et al.
(författare)
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Hierarchical molecular tagging to resolve long continuous sequences by massively parallel sequencing
- 2013
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 3, s. 1186-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Lundin, Sverker, et al.
(författare)
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Increased Throughput by Parallelization of Library Preparation for Massive Sequencing
- 2010
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:3, s. e10029-
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Lundin, Sverker, 1982-
(författare)
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Methods to Prepare DNA for Efficient Massive Sequencing
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 5. |
- Meletis, Konstantinos, et al.
(författare)
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p53 suppresses the self-renewal of adult neural stem cells
- 2006
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Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 133:2, s. 363-369
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Tidskriftsartikel (refereegranskat)abstract
- There is increasing evidence that tumors are heterogeneous and that a subset of cells act as cancer stem cells. Several proto-oncogenes and tumor suppressors control key aspects of stem cell function, suggesting that similar mechanisms control normal and cancer stem cell properties. We show here that the prototypical tumor suppressor p53, which plays an important role in brain tumor initiation and growth, is expressed in the neural stem cell lineage in the adult brain. p53 negatively regulates proliferation and survival, and thereby self-renewal, of neural stem cells. Analysis of the neural stem cell transcriptome identified the dysregulation of several cell cycle regulators in the absence of p53, most notably a pronounced downregulation of p21 expression. These data implicate p53 as a suppressor of tissue and cancer stem cell self-renewal.
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| 6. |
- Pettersson, Erik, et al.
(författare)
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Allelotyping by massively parallel pyrosequencing of SNP-carrying trinucleotide threads
- 2008
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Ingår i: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 29:2, s. 323-329
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Tidskriftsartikel (refereegranskat)abstract
- Here we present an approach for allelotyping combining the multiplexing features of the trinucleotide threading (TnT) method with pooling of genomic DNA and massively parallel pyrosequencing, enabling reliable allele frequency estimation in large cohorts. The approach offers several benefits as compared to array-based methods and allows undertaking highly complex studies without compromising accuracy, while keeping the workload to a minimum. This proof-of-concept study involves formation of trinucleotide threads, targeting a total of 147 single-nucleotide polymorphisms (SNPs) related to obesity and cancer, for multiplex amplification and allele extraction from a pool of 462 genomes, followed by massively parallel pyrosequencing. Approximately 177k reads were approved, identified, and assigned to SNP-carrying threads rendering representative allele frequencies in the cohort.
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| 7. |
- Richter, Karin, et al.
(författare)
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Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression
- 2006
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 7, s. 75-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types.Results: Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development.Conclusion: Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin.
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| 8. |
- Sievertzon, Maria, et al.
(författare)
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Epidermal growth factor (EGF) withdrawal masks gene expression differences in the study of pituitary adenylate cyclase-activating polypeptide (PACAP) activation of primary neural stem cell proliferation
- 2005
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Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 6, s. 55-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The recently discovered adult neural stem cells, which maintain continuous generation of new neuronal and glial cells throughout adulthood, are a promising and expandable source of cells for use in cell replacement therapies within the central nervous system. These cells could either be induced to proliferate and differentiate endogenously, or expanded and differentiated in culture before being transplanted into the damaged site of the brain. In order to achieve these goals effective strategies to isolate, expand and differentiate neural stem cells into the desired specific phenotypes must be developed. However, little is known as yet about the factors and mechanisms influencing these processes. It has recently been reported that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neural stem cell proliferation both in vivo and in vitro. Results: We used cDNA microarrays with the aim of analysing the transcriptional changes underlying PACAP induced proliferation of neural stem cells. The primary neural stem/progenitor cells used were neurospheres, generated from the lateral ventricle wall of the adult mouse brain. The results were compared to both differentiation and proliferation controls, which revealed an unexpected and significant differential expression relating to withdrawal of epidermal growth factor (EGF) from the neurosphere growth medium. The effect of EGF removal was so pronounced that it masked the changes in gene expression patterns produced by the addition of PACAP. Conclusion: Experimental models aiming at transcriptional analysis of induced proliferation in primary neural stem cells need to take into consideration the significant effect on transcription caused by removal of EGF. Alternatively, EGF-free culture conditions need to be developed.
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| 9. |
- Sievertzon, Maria, et al.
(författare)
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Transcriptome analysis in primary neural stem cells using a tag cDNA amplification method
- 2005
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Ingår i: BMC Neuroscience. - : Springer Science and Business Media LLC. - 1471-2202. ; 6:28, s. 13-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Neural stem cells ( NSCs) can be isolated from the adult mammalian brain and expanded in culture, in the form of cellular aggregates called neurospheres. Neurospheres provide an in vitro model for studying NSC behaviour and give information on the factors and mechanisms that govern their proliferation and differentiation. They are also a promising source for cell replacement therapies of the central nervous system. Neurospheres are complex structures consisting of several cell types of varying degrees of differentiation. One way of characterising neurospheres is to analyse their gene expression profiles. The value of such studies is however uncertain since they are heterogeneous structures and different populations of neurospheres may vary significantly in their gene expression. Results: To address this issue, we have used cDNA microarrays and a recently reported tag cDNA amplification method to analyse the gene expression profiles of neurospheres originating from separate isolations of the lateral ventricle wall of adult mice and passaged to varying degrees. Separate isolations as well as consecutive passages yield a high variability in gene expression while parallel cultures yield the lowest variability. Conclusions: We demonstrate a low technical amplification variability using the employed amplification strategy and conclude that neurospheres from the same isolation and passage are sufficiently similar to be used for comparative gene expression analysis.
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| 10. |
- Wirta, Valtteri, 1977-
(författare)
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Mining the transcriptome - methods and applications
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods. The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal. This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype. To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank.
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