Publications by authors named "Paul H Dear"

34 Publications

Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Nucleic Acids Res 2020 12;48(22):e132

Base 4 Innovation Ltd, Broers Building, JJ Thomson Avenue, Cambridge CB3 0FA, UK.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736801PMC
December 2020

Towards accurate exclusion of neonatal bacterial meningitis: a feasibility study of a novel 16S rDNA PCR assay.

BMC Infect Dis 2020 Jun 22;20(1):441. Epub 2020 Jun 22.

Mote Research Ltd., Babraham, Cambridge, UK.

Background: PCRctic is an innovative assay based on 16S rDNA PCR technology that has been designed to detect a single intact bacterium in a specimen of cerebro-spinal fluid (CSF). The assay's potential for accurate, fast and inexpensive discrimination of bacteria-free CSF makes it an ideal adjunct for confident exclusion of bacterial meningitis in newborn babies where the negative predictive value of bacterial culture is poor. This study aimed to stress-test and optimize PCRctic in the "field conditions" to attain a clinically useful level of specificity.

Methods: The specificity of PCRctic was evaluated in CSF obtained from newborn babies investigated for meningitis on a tertiary neonatal unit. Following an interim analysis, the method of skin antisepsis was changed to increase bactericidal effect, and snap-top tubes (Eppendorf™) replaced standard universal containers for collection of CSF to reduce environmental contamination.

Results: The assay's specificity was 90.5% in CSF collected into the snap-top tubes - up from 60% in CSF in the universal containers. The method of skin antisepsis had no effect on the specificity. All CSF cultures were negative and no clinical cases of neonatal bacterial meningitis occurred during the study.

Conclusions: A simple and inexpensive optimization of CSF collection resulted in a high specificity output. The low prevalence of neonatal bacterial meningitis means that a large multi-centre study will be required to validate the assay's sensitivity and its negative predictive value.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12879-020-05160-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310343PMC
June 2020

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

Nucleic Acids Res 2019 09;47(17):e101

Base4 Innovation Ltd, Broers Building, 21 JJ Thomson Avenue, Cambridge CB3 0FA, UK.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753480PMC
September 2019

Perigone Lobe Transcriptome Analysis Provides Insights into Rafflesia cantleyi Flower Development.

PLoS One 2016 15;11(12):e0167958. Epub 2016 Dec 15.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia.

Rafflesia is a biologically enigmatic species that is very rare in occurrence and possesses an extraordinary morphology. This parasitic plant produces a gigantic flower up to one metre in diameter with no leaves, stem or roots. However, little is known about the floral biology of this species especially at the molecular level. In an effort to address this issue, we have generated and characterised the transcriptome of the Rafflesia cantleyi flower, and performed a comparison with the transcriptome of its floral bud to predict genes that are expressed and regulated during flower development. Approximately 40 million sequencing reads were generated and assembled de novo into 18,053 transcripts with an average length of 641 bp. Of these, more than 79% of the transcripts had significant matches to annotated sequences in the public protein database. A total of 11,756 and 7,891 transcripts were assigned to Gene Ontology categories and clusters of orthologous groups respectively. In addition, 6,019 transcripts could be mapped to 129 pathways in Kyoto Encyclopaedia of Genes and Genomes Pathway database. Digital abundance analysis identified 52 transcripts with very high expression in the flower transcriptome of R. cantleyi. Subsequently, analysis of differential expression between developing flower and the floral bud revealed a set of 105 transcripts with potential role in flower development. Our work presents a deep transcriptome resource analysis for the developing flower of R. cantleyi. Genes potentially involved in the growth and development of the R. cantleyi flower were identified and provide insights into biological processes that occur during flower development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167958PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5158018PMC
July 2017

Structure of the germline genome of and relationship to the massively rearranged somatic genome.

Elife 2016 11 28;5. Epub 2016 Nov 28.

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, United States.

The germline genome of the binucleated ciliate undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare 's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.19090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5182062PMC
November 2016

Comparative Genome Analysis Reveals Divergent Genome Size Evolution in a Carnivorous Plant Genus.

Plant Genome 2015 Nov;8(3):eplantgenome2015.04.0021

Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466, Gatersleben, Germany.

The C-value paradox remains incompletely resolved after >40 yr and is exemplified by 2,350-fold variation in genome sizes of flowering plants. The carnivorous Lentibulariaceae genus Genlisea, displaying a 25-fold range of genome sizes, is a promising subject to study mechanisms and consequences of evolutionary genome size variation. Applying genomic, phylogenetic, and cytogenetic approaches, we uncovered bidirectional genome size evolution within the genus Genlisea. The Genlisea nigrocaulis Steyerm. genome (86 Mbp) has probably shrunk by retroelement silencing and deletion-biased double-strand break (DSB) repair, from an ancestral size of 400 to 800 Mbp to become one of the smallest among flowering plants. The G. hispidula Stapf genome has expanded by whole-genome duplication (WGD) and retrotransposition to 1550 Mbp. Genlisea hispidula became allotetraploid after the split from the G. nigrocaulis clade ∼29 Ma. Genlisea pygmaea A. St.-Hil. (179 Mbp), a close relative of G. nigrocaulis, proved to be a recent (auto)tetraploid. Our analyses suggest a common ancestor of the genus Genlisea with an intermediate 1C value (400-800 Mbp) and subsequent rapid genome size evolution in opposite directions. Many abundant repeats of the larger genome are absent in the smaller, casting doubt on their functionality for the organism, while recurrent WGD seems to safeguard against the loss of essential elements in the face of genome shrinkage. We cannot identify any consistent differences in habitat or life strategy that correlate with genome size changes, raising the possibility that these changes may be selectively neutral.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3835/plantgenome2015.04.0021DOI Listing
November 2015

Genomic analysis of the causative agents of coccidiosis in domestic chickens.

Genome Res 2014 Oct 11;24(10):1676-85. Epub 2014 Jul 11.

Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom;

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.168955.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199364PMC
October 2014

IRS2 is a candidate driver oncogene on 13q34 in colorectal cancer.

Int J Exp Pathol 2013 Jun 18;94(3):203-11. Epub 2013 Apr 18.

MRC Laboratory of Molecular Biology, Cambridge, UK.

Copy number alterations are frequently found in colorectal cancer (CRC), and recurrent gains or losses are likely to correspond to regions harbouring genes that promote or impede carcinogenesis respectively. Gain of chromosome 13q is common in CRC but, because the region of gain is frequently large, identification of the driver gene(s) has hitherto proved difficult. We used array comparative genomic hybridization to analyse 124 primary CRCs, demonstrating that 13q34 is a region of gain in 35% of CRCs, with focal gains in 4% and amplification in a further 1.6% of cases. To reduce the number of potential driver genes to consider, it was necessary to refine the boundaries of the narrowest copy number changes seen in this series and hence define the minimal copy region (MCR). This was performed using molecular copy-number counting, identifying IRS2 as the only complete gene, and therefore the likely driver oncogene, within the refined MCR. Analysis of available colorectal neoplasia data sets confirmed IRS2 gene gain as a common event. Furthermore, IRS2 protein and mRNA expression in colorectal neoplasia was assessed and was positively correlated with progression from normal through adenoma to carcinoma. In functional in vitro experiments, we demonstrate that deregulated expression of IRS2 activates the oncogenic PI3 kinase pathway and increases cell adhesion, both characteristics of invasive CRC cells. Together, these data identify IRS2 as a likely driver oncogene in the prevalent 13q34 region of gain/amplification and suggest that IRS2 over-expression may provide an additional mechanism of PI3 kinase pathway activation in CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/iep.12021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664965PMC
June 2013

Digital PCR strategies in the development and analysis of molecular biomarkers for personalized medicine.

Methods 2013 Jan 19;59(1):101-7. Epub 2012 Aug 19.

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom.

The efficient delivery of personalized medicine is a key goal of healthcare over the next decade. It is likely that PCR strategies will play an important role in the delivery of this goal. Digital PCR has certain advantages over more traditional PCR protocols. In this article we will discuss the current status of digital PCR, highlighting its advantages and focusing on how it can be utilized in biomarker development and analysis, including the use of individualized biomarkers. We will explore recent developments in this field including examples of how digital PCR may integrate with next generation sequencing to deliver truly personalized medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2012.08.001DOI Listing
January 2013

Insights into the genome structure and copy-number variation of Eimeria tenella.

BMC Genomics 2012 Aug 13;13:389. Epub 2012 Aug 13.

School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor DE, Malaysia.

Background: Eimeria is a genus of parasites in the same phylum (Apicomplexa) as human parasites such as Toxoplasma, Cryptosporidium and the malaria parasite Plasmodium. As an apicomplexan whose life-cycle involves a single host, Eimeria is a convenient model for understanding this group of organisms. Although the genomes of the Apicomplexa are diverse, that of Eimeria is unique in being composed of large alternating blocks of sequence with very different characteristics - an arrangement seen in no other organism. This arrangement has impeded efforts to fully sequence the genome of Eimeria, which remains the last of the major apicomplexans to be fully analyzed. In order to increase the value of the genome sequence data and aid in the effort to gain a better understanding of the Eimeria tenella genome, we constructed a whole genome map for the parasite.

Results: A total of 1245 contigs representing 70.0% of the whole genome assembly sequences (Wellcome Trust Sanger Institute) were selected and subjected to marker selection. Subsequently, 2482 HAPPY markers were developed and typed. Of these, 795 were considered as usable markers, and utilized in the construction of a HAPPY map. Markers developed from chromosomally-assigned genes were then integrated into the HAPPY map and this aided the assignment of a number of linkage groups to their respective chromosomes. BAC-end sequences and contigs from whole genome sequencing were also integrated to improve and validate the HAPPY map. This resulted in an integrated HAPPY map consisting of 60 linkage groups that covers approximately half of the estimated 60 Mb genome. Further analysis suggests that the segmental organization first seen in Chromosome 1 is present throughout the genome, with repeat-poor (P) regions alternating with repeat-rich (R) regions. Evidence of copy-number variation between strains was also uncovered.

Conclusions: This paper describes the application of a whole genome mapping method to improve the assembly of the genome of E. tenella from shotgun data, and to help reveal its overall structure. A preliminary assessment of copy-number variation (extra or missing copies of genomic segments) between strains of E. tenella was also carried out. The emerging picture is of a very unusual genome architecture displaying inter-strain copy-number variation. We suggest that these features may be related to the known ability of this parasite to rapidly develop drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2164-13-389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3505466PMC
August 2012

Numts help to reconstruct the demographic history of the ocellated lizard (Lacerta lepida) in a secondary contact zone.

Mol Ecol 2012 Feb 4;21(4):1005-18. Epub 2012 Jan 4.

School of Biological Sciences, University of East Anglia, Norwich NR4 7J, UK.

In northwestern Iberia, two largely allopatric Lacerta lepida mitochondrial lineages occur, L5 occurring to the south of Douro River and L3 to the north, with a zone of putative secondary contact in the region of the Douro River valley. Cytochrome b sequence chromatograms with polymorphisms at nucleotide sites diagnostic for the two lineages were detected in individuals in the region of the Douro River and further north within the range of L3. We show that these polymorphisms are caused by the presence of four different numts (I-IV) co-occurring with the L3 genome, together with low levels of heteroplasmy. Two of the numts (I and II) are similar to the mitochondrial genome of L5 but are quite divergent from the mitochondrial genome of L3 where they occur. We show that these numts are derived from the mitochondrial genome of L5 and were incorporated in L3 through hybridization at the time of secondary contact between the lineages. The additional incidence of these numts to the north of the putative contact zone is consistent with an earlier postglacial northward range expansion of L5, preceding that of L3. We show that genetic exchange between the lineages responsible for the origin of these numts in L3 after secondary contact occurred prior to, or coincident with, the northward expansion of L3. This study shows that, in the context of phylogeographic analysis, numts can provide evidence for past demographic events and can be useful tools for the reconstruction of complex evolutionary histories.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-294X.2011.05422.xDOI Listing
February 2012

Single-molecule analysis of genome rearrangements in cancer.

Nucleic Acids Res 2011 Jul 27;39(13):e85. Epub 2011 Apr 27.

Hutchison/MRC Research Centre and Department of Pathology, University of Cambridge, Hills Road, Cambridge, CB2 0XZ, UK.

Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkr227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3141271PMC
July 2011

Genomic evidence of pre-invasive clonal expansion, dispersal and progression in bronchial dysplasia.

J Pathol 2011 Jun 19;224(2):153-9. Epub 2011 Apr 19.

MRC Laboratory of Molecular Biology, Cambridge, UK.

The term 'field cancerization' is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.2887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3378694PMC
June 2011

Progressive 3q amplification consistently targets SOX2 in preinvasive squamous lung cancer.

Am J Respir Crit Care Med 2010 Jul 18;182(1):83-91. Epub 2010 Mar 18.

Centre for Respiratory Research, Royal Free and University College Medical School, London, United Kingdom.

Rationale: Amplification of distal 3q is the most common genomic aberration in squamous lung cancer (SQC). SQC develops in a multistage progression from normal bronchial epithelium through dysplasia to invasive disease. Identifying the key driver events in the early pathogenesis of SQC will facilitate the search for predictive molecular biomarkers and the identification of novel molecular targets for chemoprevention and therapeutic strategies. For technical reasons, previous attempts to analyze 3q amplification in preinvasive lesions have focused on small numbers of predetermined candidate loci rather than an unbiased survey of copy-number variation.

Objectives: To perform a detailed analysis of the 3q amplicon in bronchial dysplasia of different histological grades.

Methods: We use molecular copy-number counting (MCC) to analyze the structure of chromosome 3 in 19 preinvasive bronchial biopsy specimens from 15 patients and sequential biopsy specimens from 3 individuals.

Measurements And Main Results: We demonstrate that no low-grade lesions, but all high-grade lesions, have 3q amplification. None of seven low-grade lesions progressed clinically, whereas 8 of 10 patients with high-grade disease progressed to cancer. We identify a minimum commonly amplified region on chromosome 3 consisting of 17 genes, including 2 known oncogenes, SOX2 and PIK3CA. We confirm that both genes are amplified in all high-grade dysplastic lesions tested. We further demonstrate, in three individuals, that the clinical progression of high-grade preinvasive disease is associated with incremental amplification of SOX2, suggesting this promotes malignant progression.

Conclusions: These findings demonstrate progressive 3q amplification in the evolution of preinvasive SQC and implicate SOX2 as a key target of this dynamic process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1164/rccm.201001-0005OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2902760PMC
July 2010

BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping.

PLoS One 2010 Feb 8;5(2):e9089. Epub 2010 Feb 8.

Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom.

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical "contig" maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning approximately 10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual "BAC-HAPPY-mapping" to convert BAC landing data into BAC linkage contigs is possible.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009089PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2816996PMC
February 2010

Single-molecule genomics.

J Pathol 2010 Jan;220(2):297-306

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

The term 'single-molecule genomics' (SMG) describes a group of molecular methods in which single molecules are detected or sequenced. The focus on the analysis of individual molecules distinguishes these techniques from more traditional methods, in which template DNA is cloned or PCR-amplified prior to analysis. Although technically challenging, the analysis of single molecules has the potential to play a major role in the delivery of truly personalized medicine. The two main subgroups of SMG methods are single-molecule digital PCR and single-molecule sequencing. Single-molecule PCR has a number of advantages over competing technologies, including improved detection of rare genetic variants and more precise analysis of copy-number variation, and is more easily adapted to the often small amount of material that is available in clinical samples. Single-molecule sequencing refers to a number of different methods that are mainly still in development but have the potential to make a huge impact on personalized medicine in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.2647DOI Listing
January 2010

Copy-number variation: the end of the human genome?

Authors:
Paul H Dear

Trends Biotechnol 2009 Aug 1;27(8):448-54. Epub 2009 Jul 1.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB20QH, UK.

Copy-number variation (CNV)--the presence of additional or missing segments of chromosomes in some individuals--has been found to be abundant in humans and adds another dimension of variation to the genome. Copy-number variants have already been associated with some diseases and disease susceptibilities and are likely to prove as significant as sequence polymorphisms in this respect. Changes in copy number of parts of the genome are known to be a feature of many cancers, and their analysis is expected to reveal genes involved in carcinogenesis. This article will present a somewhat biased and occasionally speculative discussion of the current and future significance of CNV with a particular focus on the potential of molecular copy-number counting in the analysis of small, damaged or heterogeneous samples.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tibtech.2009.05.003DOI Listing
August 2009

Mitochondrial genomes reveal an explosive radiation of extinct and extant bears near the Miocene-Pliocene boundary.

BMC Evol Biol 2008 Jul 28;8:220. Epub 2008 Jul 28.

Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany.

Background: Despite being one of the most studied families within the Carnivora, the phylogenetic relationships among the members of the bear family (Ursidae) have long remained unclear. Widely divergent topologies have been suggested based on various data sets and methods.

Results: We present a fully resolved phylogeny for ursids based on ten complete mitochondrial genome sequences from all eight living and two recently extinct bear species, the European cave bear (Ursus spelaeus) and the American giant short-faced bear (Arctodus simus). The mitogenomic data yield a well-resolved topology for ursids, with the sloth bear at the basal position within the genus Ursus. The sun bear is the sister taxon to both the American and Asian black bears, and this clade is the sister clade of cave bear, brown bear and polar bear confirming a recent study on bear mitochondrial genomes.

Conclusion: Sequences from extinct bears represent the third and fourth Pleistocene species for which complete mitochondrial genomes have been sequenced. Moreover, the cave bear specimen demonstrates that mitogenomic studies can be applied to Pleistocene fossils that have not been preserved in permafrost, and therefore have a broad application within ancient DNA research. Molecular dating of the mtDNA divergence times suggests a rapid radiation of bears in both the Old and New Worlds around 5 million years ago, at the Miocene-Pliocene boundary. This coincides with major global changes, such as the Messinian crisis and the first opening of the Bering Strait, and suggests a global influence of such events on species radiations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1471-2148-8-220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2518930PMC
July 2008

Real-time PCRs and fingerprinting assays for the detection and characterization of Salmonella Genomic Island-1 encoding multidrug resistance: application to 445 European isolates of Salmonella, Escherichia coli, Shigella, and Proteus.

Microb Drug Resist 2008 Jun;14(2):79-92

Health Protection Agency, Centre for Infections, London, United Kingdom.

Salmonella Genomic Island-1 (SGI-1) harbors a cluster of genes encoding multidrug resistance (MDR). SGI-1 is horizontally transmissible and is therefore of significant public health concern. This study presents two novel realtime PCRs detecting three SGI-1 protein-coding genes and a SGI-1 fingerprinting assay. These assays were applied to 445 European enterobacterial isolates. Results from real-time PCRs were comparable to those obtained from gelbased PCRs used for the detection of SGI-1, but were rapid to perform and suitable for large-scale screening. Furthermore, real-time PCRs also detected SGI-1 even when only part of the island was present in bacterial isolates. No trace of SGI-1 was detected in isolates other than Salmonella enterica. The fingerprints showed that regions of SGI-1 outside the MDR region exhibited genomic variations between isolates. In conclusion, the realtime PCRs described here are suitable for the detection of SGI-1 in bacterial isolates. Further studies are necessary to elucidate divergence in its non-MDR region.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/mdr.2008.0812DOI Listing
June 2008

Multiplex amplification of ancient DNA.

Nat Protoc 2006 ;1(2):720-8

Molecular Biochemistry, Institute of Biochemistry, Medical Faculty, University of Leipzig, Johannisallee 30, 04103 Leipzig, Germany.

This method is designed to assemble long, continuous DNA sequences using minimal amounts of fragmented ancient DNA as template. This is achieved by a two-step approach. In the first step, multiple fragments are simultaneously amplified in a single multiplex reaction. Subsequently, each of the generated fragments is amplified individually using a single primer pair, in a standard simplex (monoplex) PCR. The ability to amplify multiple fragments simultaneously in the first step allows the generation of large amounts of sequence from rare template DNA, whereas the second nested step increases specificity and decreases amplification of contaminating DNA. In contrast to current protocols using many template-consuming simplex PCRs, the method described allows amplification of several kilobases of sequence in just one reaction. It thus combines optimal template usage with a high specificity and can be performed within a day.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nprot.2006.84DOI Listing
September 2007

Sequencing and analysis of chromosome 1 of Eimeria tenella reveals a unique segmental organization.

Genome Res 2007 Mar 6;17(3):311-9. Epub 2007 Feb 6.

Malaysia Genome Institute, UKM-MTDC Smart Technology Centre, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor DE, Malaysia.

Eimeria tenella is an intracellular protozoan parasite that infects the intestinal tracts of domestic fowl and causes coccidiosis, a serious and sometimes lethal enteritis. Eimeria falls in the same phylum (Apicomplexa) as several human and animal parasites such as Cryptosporidium, Toxoplasma, and the malaria parasite, Plasmodium. Here we report the sequencing and analysis of the first chromosome of E. tenella, a chromosome believed to carry loci associated with drug resistance and known to differ between virulent and attenuated strains of the parasite. The chromosome--which appears to be representative of the genome--is gene-dense and rich in simple-sequence repeats, many of which appear to give rise to repetitive amino acid tracts in the predicted proteins. Most striking is the segmentation of the chromosome into repeat-rich regions peppered with transposon-like elements and telomere-like repeats, alternating with repeat-free regions. Predicted genes differ in character between the two types of segment, and the repeat-rich regions appear to be associated with strain-to-strain variation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.5823007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1800922PMC
March 2007

An efficient method for multi-locus molecular haplotyping.

Nucleic Acids Res 2007 7;35(1):e6. Epub 2006 Dec 7.

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

Many methods exist for genotyping--revealing which alleles an individual carries at different genetic loci. A harder problem is haplotyping--determining which alleles lie on each of the two homologous chromosomes in a diploid individual. Conventional approaches to haplotyping require the use of several generations to reconstruct haplotypes within a pedigree, or use statistical methods to estimate the prevalence of different haplotypes in a population. Several molecular haplotyping methods have been proposed, but have been limited to small numbers of loci, usually over short distances. Here we demonstrate a method which allows rapid molecular haplotyping of many loci over long distances. The method requires no more genotypings than pedigree methods, but requires no family material. It relies on a procedure to identify and genotype single DNA molecules, and reconstruction of long haplotypes by a 'tiling' approach. We demonstrate this by resolving haplotypes in two regions of the human genome, harbouring 20 and 105 single-nucleotide polymorphisms, respectively. The method can be extended to reconstruct haplotypes of arbitrary complexity and length, and can make use of a variety of genotyping platforms. We also argue that this method is applicable in situations which are intractable to conventional approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkl742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1802573PMC
February 2007

HAPPY mapping in a plant genome: reconstruction and analysis of a high-resolution physical map of a 1.9 Mbp region of Arabidopsis thaliana chromosome 4.

Plant Biotechnol J 2003 Jan;1(1):23-31

MRC Laboratory of Molecular Biology, Protein and Nucleic Acid Chemistry Division, Cambridge CB2 2QH, UK.

HAPPY mapping is an in vitro approach for defining the order and spacing of DNA markers directly on native genomic DNA. This cloning-free technique is based on analysing the segregation of markers amplified from high molecular weight genomic DNA which has been broken randomly and 'segregated' by limiting dilution into subhaploid samples. It is a uniquely versatile tool, allowing for the construction of genome maps with flexible ranges and resolutions. Moreover, it is applicable to plant genomes, for which many of the techniques pioneered in animal genomes are inapplicable or inappropriate. We report here its demonstration in a plant genome by reconstructing the physical map of a 1.9 Mbp region around the FCA locus of Arabidopsis thaliana. The resulting map, spanning around 10% of chromosome 4, is in excellent agreement with the DNA sequence and has a mean marker spacing of 16 kbp. We argue that HAPPY maps of any required resolution can be made immediately and with relatively little effort for most plant species and, furthermore, that such maps can greatly aid the construction of regional or genome-wide physical maps.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1046/j.1467-7652.2003.00001.xDOI Listing
January 2003

Use of HAPPY mapping for the higher order assembly of the Tetrahymena genome.

Genomics 2006 Oct 19;88(4):443-51. Epub 2006 Jun 19.

Department of Molecular, Cellular, and Developmental Biology, University of California at Santa Barbara, Santa Barbara, CA 93106, USA.

Tetrahymena thermophila is the best studied of the ciliates, a diversified and successful lineage of eukaryotic protists. Mirroring the way in which many metazoans partition their germ line and soma into distinct cell types, ciliates separate germ line and soma into two distinct nuclei in a single cell. The diploid, transcriptionally silent micronucleus undergoes meiosis and fertilization during sexual reproduction and determines the genotype of the progeny; in contrast, the expressed macronucleus contains many copies of hundreds of small chromosomes, determines the cell's phenotype, and is inherited only through vegetative reproduction. Here we demonstrate the power of HAPPY physical mapping to aid the complete assembly of T. thermophila macronuclear chromosomes from shotgun sequence scaffolds. The finished genome, one of only two ciliate genomes shotgun sequenced, will shed valuable additional light upon the biology of this extraordinary, diverse, and, from a genomics standpoint, as yet largely unexplored evolutionary branch of eukaryotes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ygeno.2006.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169840PMC
October 2006

Interrogation of genomes by molecular copy-number counting (MCC).

Nat Methods 2006 Jun;3(6):447-53

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the breakpoint without recourse to genomic libraries. The analysis also revealed additional cryptic chromosomal changes close to the translocation junction. MCC is a fast and flexible method for characterizing a wide range of chromosomal aberrations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth880DOI Listing
June 2006

Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae.

Nature 2006 Feb 18;439(7077):724-7. Epub 2005 Dec 18.

Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6, D-04103 Leipzig, Germany.

In studying the genomes of extinct species, two principal limitations are typically the small quantities of endogenous ancient DNA and its degraded condition, even though products of up to 1,600 base pairs (bp) have been amplified in rare cases. Using small overlapping polymerase chain reaction products, longer stretches of sequences or even whole mitochondrial genomes can be reconstructed, but this approach is limited by the number of amplifications that can be performed from rare samples. Thus, even from well-studied Pleistocene species such as mammoths, ground sloths and cave bears, no DNA sequences of more than about 1,000 bp have been reconstructed. Here we report the complete mitochondrial genome sequence of the Pleistocene woolly mammoth Mammuthus primigenius. We used about 200 mg of bone and a new approach that allows the simultaneous retrieval of multiple sequences from small amounts of degraded DNA. Our phylogenetic analyses show that the mammoth was more closely related to the Asian than to the African elephant. However, the divergence of mammoth, African and Asian elephants occurred over a short time, corresponding to only about 7% of the total length of the phylogenetic tree for the three evolutionary lineages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature04432DOI Listing
February 2006

The genome of Cryptosporidium hominis.

Nature 2004 Oct;431(7012):1107-12

Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23284-2030, USA.

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa--protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome approximately 9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature02977DOI Listing
October 2004

One by one: Single molecule tools for genomics.

Authors:
Paul H Dear

Brief Funct Genomic Proteomic 2003 Jan;1(4):397-416

MRC Laboratory of Molecular Biology, Cambridge, UK.

Much of the effort in any genomics programme arises from the need to generate and purify large numbers of identical molecules, since most analytical tools rely on the analysis of bulk DNA. Biological steps such as bacterial cloning--commonly used to prepare bulk samples of defined DNA fragments--are capricious and introduce their own restrictions and distortions. The analysis of single molecules, either directly or by in vitro enzymatic amplification, makes possible the examination of native genomic DNA without the complications and restrictions of biological propagation. Techniques already exist for the in vitro propagation of genomic fragments and for genome mapping, and offer the advantages of speed, flexibility and predictable behaviour. Single molecule sequencing, for which many approaches are being developed, is more challenging, but offers even greater rewards in terms of throughput and read length.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/bfgp/1.4.397DOI Listing
January 2003

The Eimeria genome projects: a sequence of events.

Trends Parasitol 2004 May;20(5):199-201

Institute for Animal Health, Compton Laboratory, Compton, Nr Newbury, Berkshire RG20 7NN, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.pt.2004.02.005DOI Listing
May 2004

Complete genome sequence of the apicomplexan, Cryptosporidium parvum.

Science 2004 Apr 25;304(5669):441-5. Epub 2004 Mar 25.

Department of Veterinary and Biomedical Science, College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA.

The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals, and causes an unrelenting infection in immunocompromised individuals such as AIDS patients. We report the complete genome sequence of C. parvum, type II isolate. Genome analysis identifies extremely streamlined metabolic pathways and a reliance on the host for nutrients. In contrast to Plasmodium and Toxoplasma, the parasite lacks an apicoplast and its genome, and possesses a degenerate mitochondrion that has lost its genome. Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected. Elucidation of the core metabolism, including enzymes with high similarities to bacterial and plant counterparts, opens new avenues for drug development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1094786DOI Listing
April 2004
-->