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Technical tips, new applications, and product highlights from Epicentre Biotechnologies.
While Epicentre’s novel Nextera™ technology is revolutionizing next-generation sequencing library preparation, many laboratories are still using older methods of creating genomic DNA libraries for next-generation sequencing. A recent study (Cheung et al.*) of transcriptional regulation mediated by trimethylated histone H3K4 used ChIP-Seq analysis in samples obtained from the human prefrontal cortex.Preparation of the ChIP-Seq libraries involved several Epicentre products: the End-It™ DNA E........ Read more »
Cheung, I. et al. (2010) Developmental regulation and individual differences of neuronal H3K4me3 epigenomes in the prefrontal cortex. Proceedings of the National Academy of Sciences, 107(19), 8824-8829. DOI: 10.1073/pnas.1001702107
Helicobacter pylori infects about 50% of the human population, and is implicated in inflammation, ulcers, and gastric cancer. Sharma et al.* used a novel approach--differential RNA-Seq (dRNA-Seq)--that selects for the 5’ ends of primary transcripts to construct a genome-wide map of transcriptional start sites (TSS) and operons.... Read more »
Sharma, C. et al. (2010) The primary transcriptome of the major human pathogen Helicobacter pylori. Nature, 464(7286), 250-255. DOI: 10.1038/nature08756
With the advent of next-generation sequencing techniques, genomic cloning has taken on a new role in de novo and reference-genome sequencing. Previously, Kitzman et al.1 generated massive amounts of shotgun sequence data from a human library by sequencing 120 pools of CopyControl™ fosmid clones using Nextera™ DNA library preparation technology. This method generated sequencing data of sufficient depth to assemble a complete haplotype-resolved genome for the individual being studied.
However........ Read more »
Kitzman, J. et al. (2010) Haplotype-resolved genome sequencing of a Gujarati Indian individual. Nature Biotechnology, 29(1), 59-63. DOI: 10.1038/nbt.1740
Naka, H. et al. (2011) Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii. Infection and Immunity, 79(7), 2889-2900. DOI: 10.1128/IAI.05138-11
Acinetobacter baumanii is a pathogenic bacterium that has been demonstrated to cause pneumonia, skin infections, and secondary meningitis, predominantly in a health-care facility setting. Its ability to form biofilms on inert surfaces is instrumental in creating reservoirs for opportunistic infection.... Read more »
Jacobs, A. et al. (2010) Inactivation of Phospholipase D Diminishes Acinetobacter baumannii Pathogenesis. Infection and Immunity, 78(5), 1952-1962. DOI: 10.1128/IAI.00889-09
Esophageal cancer is currently the eighth most common human cancer, with esophageal squamous cell carcinoma (ESCC) being the most common subtype. Tobacco and alcohol use are the most prevalent causes of ESCC; however, limited evidence suggests that infectious agents--in particular, human papillomavirus (HPV)--are linked to ESCC. Antonsson et al. recently analyzed HPV prevalence and lifestyle factors in ESCC patients. Archived tumor samples from a nationwide cohort of 222 ESCC patients in Austral........ Read more »
Antonsson, A. et al. (2010) High-Risk Human Papillomavirus in Esophageal Squamous Cell Carcinoma. Cancer Epidemiol Biomarkers Prev, 19(8), 2080-2087. DOI: 10.1158/1055-9965.EPI-10-0033
In a recent functional genomics study, Sommer et al. cite the use of the CopyControl™ Fosmid Library Production Kit to create a library from plant biomass DNA. Plant biomass is being explored for use in new biofuel development, in an effort to discover genetic functionalities that will allow growth improvement in key microbes by overcoming toxic/inhibitory compounds that are byproducts of biofuel conversions. Clones harboring these fosmids were tested against seven known growth inhibitors from........ Read more »
Sommer, M. et al. (2010) A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion. Molecular Systems Biology. DOI: 10.1038/msb.2010.16
In a recent publication, Lamm et al. demonstrated the use of Epicentre’s CircLigase ssDNA Ligase for template generation in high-throughput RNA-Seq applications. The authors used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of Caenorhabditis elegans. The CircLigase method relies on RNA fragmentation, poly(A) tailing, and oligo(dT)-based hybrid capture to generate a cDNA that can be recircularized and PCR-amplified for hig........ Read more »
Lamm, A. et al. (2010) Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome. Genome Research. DOI: 10.1101/gr.108845.110
Over the years, we’ve had many inquiries about using the EZ-Tn5™ Transposomes on biologically interesting but difficult-to-mutate bacteria--usually Gram-positive bacteria that have poorly understood genetics and are difficult to transform with foreign DNA. As time has progressed, some of the difficulties of using EZ-Tn5 Transposomes have been overcome. An example of such success was recently reported by Vidal et al.* regarding the use of a custom EZ-Tn5 Transposome that confers tetracycline ........ Read more »
Vidal, J. et al. (2009) Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13. PLoS ONE, 4(7). DOI: 10.1371/journal.pone.0006232
The CopyControl™ Fosmid Library Production Kit has established itself as the molecular tool of choice in studying many facets of environmental metagenomics. Donato et al. at the University of Wisconsin-Madison explored several antibiotic resistance genes from soil microbes in an apple orchard. The study focused on Streptomyces bacteria, long known to be a reservoir of multiple antimicrobial resistance markers. Older techniques, such as cultivation of microbes from soil and determining resista........ Read more »
Donato, J. et al. (2010) Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins. Applied and Environmental Microbiology, 76(13), 4396-4401. DOI: 10.1128/AEM.01763-09
A new publication from the David O’Connor laboratory at UW-Madison describes the use of Nextera™ technology to sequence whole HIV and SIV genomes.
The authors sequenced virus from an Indian rhesus macaque experimentally infected with SIVmac239 and coding regions from 11 HIV-positive patients. Overlapping RT-PCR amplicons were used to cover the virus genome sequences, and the RT-PCR-amplified genomes were simultaneously fragmented and tagged using the Nextera Roche 454-Compatible Enzyme Mix,........ Read more »
Bimber, B. et al. (2010) Whole genome characterization of HIV/SIV intra-host diversity by ultra-deep pyrosequencing. Journal of Virology. DOI: 10.1128/JVI.01378-10
Microsatellites, or simple sequence repeats (SSRs), are molecular markers that can be readily investigated for population genetic studies. Microsatellites contain tandem repeats of 1-6 bases and are usually highly polymorphic, displaying a large number of alleles. The high degree of polymorphism makes microsatellites an ideal tool for studying gene-flow.
A recent study by Pinzon et al. developed ten polymorphic microsatellite loci for a common algae (Symbiodinium fitti, type A3) to study coral-........ Read more »
Pinzón, J. et al. (2010) Microsatellite loci for Symbiodinium A3 (S. fitti) a common algal symbiont among Caribbean Acropora (stony corals) and Indo-Pacific giant clams (Tridacna). Conservation Genetics Resources, 3(1), 45-47. DOI: 10.1007/s12686-010-9283-5
Although advances in next-generation sequencing technology have replaced the need for clone libraries in many laboratories, fosmid libraries are still useful in a variety of functional genomics studies.
Xu et al.1 present the first report of a host-specific restriction system associated with S-modification of DNA (phosphorothioation), instead of methylation. The authors observed that the enteropathogenic Salmonella enterica serovar Cerro 87, which possesses S-modified DNA, restricts DNA isolate........ Read more »
Xu, T. et al. (2010) A novel host-specific restriction system associated with DNA backbone S-modification in Salmonella. Nucleic Acids Research. DOI: 10.1093/nar/gkq610
Lucker, S. et al. (2010) A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria. Proceedings of the National Academy of Sciences. DOI: 10.1073/pnas.1003860107
Ebola virus (EBOV) and Marburg virus (MARV) are related pathogens that cause hemorrhagic fevers. In many cases, viral infections are fatal. Both viruses are native to Africa where outbreaks have been occurring for decades. There is no effective therapy for the hemorrhagic fevers caused by these viruses. EBOV and MARV are in the same taxonomic family and are structurally identical; however, they elicit different antibodies. Enterlein et al.* used the AmpliScribe™ T7 High Yield Transcription Kit........ Read more »
Enterlein, S. et al. (2009) The Marburg Virus 3' Noncoding Region Structurally and Functionally Differs from That of Ebola Virus. Journal of Virology, 83(9), 4508-4519. DOI: 10.1128/JVI.02429-08
Adey et al. (in the laboratory of Jay Shendure, University of Washington) recently published a methods paper characterizing various library prep technologies for high-throughput DNA sequencing, including Epicentre’s Nextera™ technology. The publication highlights recent advances in DNA library preparation for next-generation sequencing, in order to overcome the bottleneck posed by earlier methods, i.e., labor, time, and lack of automation.
With Nextera technology, it is now possible to prep........ Read more »
Adey, A. et al. (2010) Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biology, 11(12). DOI: 10.1186/gb-2010-11-12-r119
In order to develop guidelines for clinical diagnostic tests using gene expression profiling based on qRT-PCR analyses of formalin-fixed, paraffin-embedded (FFPE) tissues, Sánchez-Navarro et al.* compared the performance of different normalization strategies in the correlation of quantitative data between fresh-frozen (FF) and FFPE tissues. A significant challenge to expression analysis of FFPE samples is the substantial degradation of RNA extracted from these tissues, resulting in a shif........ Read more »
Sánchez-Navarro, I. et al. (2010) Comparison of gene expression profiling by reverse transcription quantitative PCR between fresh frozen and formalin-fixed, paraffin-embedded breast cancer tissues. BioTechniques, 48(May 2010), 389-397. info:/10.2144/000113388
The study of copy-number variation (CNV) in humans has contributed to our understanding of genetic uniqueness, as well as disease. Until recently, it was difficult to assess the number of repeated DNA sequences in the genome. In a recent publication, researchers at the 1000 Genomes Project and collaborators have invented new methods to study and find repetitive DNA sequences in the human genome, and have found that CNVs occur in only 7%-9% of human genes. They used the new techniques to compare ........ Read more »
Sudmant, P. et al. (2010) Diversity of Human Copy Number Variation and Multicopy Genes. Science, 330(6004), 641-646. DOI: 10.1126/science.1197005
Last November, Complete Genomics published details of its third-generation sequencing platform that promises extremely high throughput at an estimated per-library cost of $4,400 for consumables.... Read more »
Drmanac, R. et al. (2009) Human Genome Sequencing Using Unchained Base Reads on Self-Assembling DNA Nanoarrays. Science, 327(5961), 78-81. DOI: 10.1126/science.1181498
In a recent publication, Kumar et al. demonstrate the power of their “miR-ID” method in expression profiling of microRNA (miRNA) molecules. The authors used a ligase-based recircularization procedure and compared T4 RNA Ligase to Epicentre’s Circligase™ II enzyme (typically used for ssDNA circularization) in recircularization of miRNAs. They found that, while both ligases were able to join the ends of standard miRNA molecules with 2’ and 3’-hydroxyl groups, only Circligase II-based r........ Read more »
Kumar, P. et al. (2010) miR-ID: A novel, circularization-based platform for detection of microRNAs. RNA, 17(2), 365-380. DOI: 10.1261/rna.2490111
Contaminated food is becoming an increasing bacteria are the most common cause of food-borne illnesses. Lambertz et al. at the National Food Administration in Finland describe a sensitive and specific real-time PCR assay to detect the food-borne pathogen Yersinia pseudotuberculosis. Traditional culture-based methods are limited by the low isolation rate of this Gram-negative bacterium in naturally contaminated samples. Using the MasterPure™ Complete DNA and RNA Purification Kit, the rese........ Read more »
Lambertz, S. et al. (2008) TaqMan-Based Real-Time PCR Method for Detection of Yersinia pseudotuberculosis in Food. Applied and Environmental Microbiology, 74(20), 6465-6469. DOI: 10.1128/AEM.01459-08
Recently, honey bee (Apis mellifera) populations in North America and in Europe have been experiencing increased annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Population loss of honey bee colonies poses grave risks to agriculture, due to the importance of these insects in pollination of food crops. Since the effect of environmental pathogens on bees has been poorly studied, the causes of CCD have not beeen well characterized. Thus, it is necessary to build a data........ Read more »
Runckel, C. et al. (2011) Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia. PLoS ONE, 6(6). DOI: 10.1371/journal.pone.0020656
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