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

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.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

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.

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

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.

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

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.

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

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.

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

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.

Single-molecule analysis of genome rearrangements in cancer.

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.

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

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.

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

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.

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

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.

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

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.

Aerotaxis Assay in Caenorhabditis elegans to Study Behavioral Plasticity.

C. elegans shows robust and reproducible behavioral responses to oxygen. Specifically, worms prefer O 2 levels of 5-10% and avoid too high or too low O 2 . Their O 2 preference is not fixed but shows plasticity depending on experience, context, or genetic background. We recently showed that this experience-dependent plasticity declines with age, providing a useful behavioral readout for studying the mechanisms of age-related decline of neural plasticity. Here, we describe a technique to visualize behavioral O 2 preference and its plasticity in C. elegans , by creating spatial gradients of [O 2 ] in a microfluidic polydimethylsiloxane (PDMS) chamber and recording the resulting spatial distribution of the animals.

Single-molecule genomics.

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.

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

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.

The genome of Cryptosporidium hominis.

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.

An efficient method for multi-locus molecular haplotyping.

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.

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

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.

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.

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.

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

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.

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

The germline genome of the binucleated ciliate Tetrahymena thermophila 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 Tetrahymena 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 Tetrahymena's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.