共查询到20条相似文献,搜索用时 15 毫秒
1.
Sébastien Rodrigue Rex R. Malmstrom Aaron M. Berlin Bruce W. Birren Matthew R. Henn Sallie W. Chisholm 《PloS one》2009,4(9)
Background
Single-cell genome sequencing has the potential to allow the in-depth exploration of the vast genetic diversity found in uncultured microbes. We used the marine cyanobacterium Prochlorococcus as a model system for addressing important challenges facing high-throughput whole genome amplification (WGA) and complete genome sequencing of individual cells.Methodology/Principal Findings
We describe a pipeline that enables single-cell WGA on hundreds of cells at a time while virtually eliminating non-target DNA from the reactions. We further developed a post-amplification normalization procedure that mitigates extreme variations in sequencing coverage associated with multiple displacement amplification (MDA), and demonstrated that the procedure increased sequencing efficiency and facilitated genome assembly. We report genome recovery as high as 99.6% with reference-guided assembly, and 95% with de novo assembly starting from a single cell. We also analyzed the impact of chimera formation during MDA on de novo assembly, and discuss strategies to minimize the presence of incorrectly joined regions in contigs.Conclusions/Significance
The methods describe in this paper will be useful for sequencing genomes of individual cells from a variety of samples. 相似文献2.
Dirk Repsilber Sabine Kern Anna Telaar Gerhard Walzl Gillian F Black Joachim Selbig Shreemanta K Parida Stefan HE Kaufmann Marc Jacobsen 《BMC bioinformatics》2010,11(1):27
Background
For heterogeneous tissues, such as blood, measurements of gene expression are confounded by relative proportions of cell types involved. Conclusions have to rely on estimation of gene expression signals for homogeneous cell populations, e.g. by applying micro-dissection, fluorescence activated cell sorting, or in-silico deconfounding. We studied feasibility and validity of a non-negative matrix decomposition algorithm using experimental gene expression data for blood and sorted cells from the same donor samples. Our objective was to optimize the algorithm regarding detection of differentially expressed genes and to enable its use for classification in the difficult scenario of reversely regulated genes. This would be of importance for the identification of candidate biomarkers in heterogeneous tissues. 相似文献3.
Zbigniew T. Czy? Martin Hoffmann Günter Schlimok Bernhard Polzer Christoph A. Klein 《PloS one》2014,9(1)
Background
Disseminated cancer cells (DCCs) and circulating tumor cells (CTCs) are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape.Methodology/Principal Findings
Using the Ampli1™ whole genome amplification (WGA) technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points.Conclusions/Significance
The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations. 相似文献4.
Background
Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1–200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen® and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR® Identifiler® panel) and N = 49 SNP (TaqMan®) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.Results
The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.Conclusion
The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan® SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR® Identifiler® panel from wgaDNA equivalent to that of gDNA.5.
Background
The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. 相似文献6.
Yohei Nishikawa Masahito Hosokawa Toru Maruyama Keisuke Yamagishi Tetsushi Mori Haruko Takeyama 《PloS one》2015,10(9)
Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 105 droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples. 相似文献
7.
Whole genome sequencing of a single Bos taurus animal for single nucleotide polymorphism discovery 总被引:1,自引:0,他引:1
Sebastian H Eck Anna Benet-Pagès Krzysztof Flisikowski Thomas Meitinger Ruedi Fries Tim M Strom 《Genome biology》2009,10(8):R82-8
Background
The majority of the 2 million bovine single nucleotide polymorphisms (SNPs) currently available in dbSNP have been identified in a single breed, Hereford cattle, during the bovine genome project. In an attempt to evaluate the variance of a second breed, we have produced a whole genome sequence at low coverage of a single Fleckvieh bull. 相似文献8.
Jean-Marc Celton Alan Christoffels Daniel J Sargent Xiangming Xu D Jasper G Rees 《BMC biology》2010,8(1):155
Background
Determining the position and order of contigs and scaffolds from a genome assembly within an organism's genome remains a technical challenge in a majority of sequencing projects. In order to exploit contemporary technologies for DNA sequencing, we developed a strategy for whole genome single nucleotide polymorphism sequencing allowing the positioning of sequence contigs onto a linkage map using the bin mapping method. 相似文献9.
Background
Proline (Pro) accumulation is a widespread response of prokaryotic and eukaryotic cells subjected to osmotic stress or dehydration. When the cells are released from stress, Pro is degraded to glutamate by Pro-dehydrogenase (ProDH) and Pyrroline-5-carboxylate dehydrogenase (P5CDH), which are both mitochondrial enzymes in eukaryotes. While P5CDH is a single copy gene in Arabidopsis, two ProDH genes have been identified in the genome. Until now, only ProDH1 (At3g30775) had been functionally characterised. 相似文献10.
11.
Massimo Pindo Silvia Vezzulli Giuseppina Coppola Dustin A Cartwright Andrey Zharkikh Riccardo Velasco Michela Troggio 《BMC plant biology》2008,8(1):1-6
Background
Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods.Results
Herein we report the first application of the SNPlex? genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay.Conclusion
Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories. 相似文献12.
Background
Evolution of the deuterostome lineage was accompanied by an increase in systematic complexity especially with regard to highly specialized tissues and organs. Based on the observation of an increased number of paralogous genes in vertebrates compared with invertebrates, two entire genome duplications (2R) were proposed during the early evolution of vertebrates. Most glycolytic enzymes occur as several copies in vertebrate genomes, which are specifically expressed in certain tissues. Therefore, the glycolytic pathway is particularly suitable for testing theories of the involvement of gene/genome duplications in enzyme evolution. 相似文献13.
14.
Background
It has been an abiding belief among geneticists that multicellular organisms’ genomes can be analyzed under the assumption that a single individual has a uniform genome in all its cells. Despite some evidence to the contrary, this belief has been used as an axiomatic assumption in most genome analysis software packages. In this paper we present observations in human whole genome data, human whole exome data and in mouse whole genome data to challenge this assumption. We show that heterogeneity is in fact ubiquitous and readily observable in ordinary Next Generation Sequencing (NGS) data.Results
Starting with the assumption that a single NGS read (or read pair) must come from one haplotype, we built a procedure for directly observing haplotypes at a local level by examining 2 or 3 adjacent single nucleotide polymorphisms (SNPs) which are close enough on the genome to be spanned by individual reads. We applied this procedure to NGS data from three different sources: whole genome of a Central European trio from the 1000 genomes project, whole genome data from laboratory-bred strains of mouse, and whole exome data from a set of patients of head and neck tumors. Thousands of loci were found in each genome where reads spanning 2 or 3 SNPs displayed more than two haplotypes, indicating that the locus is heterogeneous. We show that such loci are ubiquitous in the genome and cannot be explained by segmental duplications. We explain them on the basis of cellular heterogeneity at the genomic level. Such heterogeneous loci were found in all normal and tumor genomes examined.Conclusions
Our results highlight the need for new methods to analyze genomic variation because existing ones do not systematically consider local haplotypes. Identification of cancer somatic mutations is complicated because of tumor heterogeneity. It is further complicated if, as we show, normal tissues are also heterogeneous. Methods for biomarker discovery must consider contextual haplotype information rather than just whether a variant “is present”.Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-418) contains supplementary material, which is available to authorized users. 相似文献15.
David A Fitzpatrick Mary E Logue Jason E Stajich Geraldine Butler 《BMC evolutionary biology》2006,6(1):99-15
Background
To date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available. 相似文献16.
Bjorn Kloosterman AM Anithakumari Pierre-Yves Chibon Marian Oortwijn Gerard C van der Linden Richard GF Visser Christian WB Bachem 《BMC plant biology》2012,12(1):17
Background
With the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization. 相似文献17.
Thomas Lingner Stefanie Mühlhausen Toni Gabaldón Cedric Notredame Peter Meinicke 《BMC bioinformatics》2010,11(1):481
Background
Establishing the relationship between an organism's genome sequence and its phenotype is a fundamental challenge that remains largely unsolved. Accurately predicting microbial phenotypes solely based on genomic features will allow us to infer relevant phenotypic characteristics when the availability of a genome sequence precedes experimental characterization, a scenario that is favored by the advent of novel high-throughput and single cell sequencing techniques. 相似文献18.
19.
Background
To explore the feasibility of constructing engineered myocardial tissues (EMTs) in vivo, using polylactic acid -co-glycolic acid (PLGA) for scaffold and cardiomyocyte-like cells derived from bone marrow mesenchymal stem cells (BMMSCs) for seeded cells. 相似文献20.