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Technical tips, new applications, and product highlights from Epicentre Biotechnologies.
Umland et al. investigated essential genes for growth in Acinetobacter baumannii by generating mutants using the EZ-Tn5™ KAN-2 Transposome. In the past, other researchers have found EZ-Tn5 transposomes extremely useful in characterizing the genetics of this bacterium.
A subset of 34 mutants with unique gene disruptions that demonstrated little to no growth on ascites underwent evaluation in a rat subcutaneous abscess model, and these results established that 18 (53%) of these genes could be classed as “in vivo essential”. The putative gene products all had known biological functions, and represented potentially untested, unrecognized, or underexploited targets for antibiotics that could be useful in treating infections in a living host. These genes could be classified into five functional categories: metabolic, two-component signaling systems, DNA/RNA synthesis and regulation, protein transport, and structural. These A. baumannii in vivo essential genes overlapped poorly with the sets of essential genes from other Gram-negative bacteria in the Database of Essential Genes (DEG), including those of Acinetobacter baylyi, a closely related species.
None of the 18 in vivo essential genes identified in this study, or their putative gene products, were classed as targets for currently existing antibiotics. The findings indicate that potentially useful antimicrobials may be developed that will be effective in treating Acinetobacter (or other) infections, though the researchers state that at this time there do not appear to be any FDA-approved antimicrobials or any other drugs in the R&D pipeline that target the newly identified genes.
Umland TC et al. (2012). In Vivo-Validated Essential Genes Identified in Acinetobacter baumannii by Using Human Ascites Overlap Poorly with Essential Genes Detected on Laboratory Media. mBio, 3 (4) PMID: 22911967
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Umland TC et al. (2012) In Vivo-Validated Essential Genes Identified in Acinetobacter baumannii by Using Human Ascites Overlap Poorly with Essential Genes Detected on Laboratory Media. mBio, 3(4). PMID: 22911967
(See Part 1)
A large archive of FFPE tumor samples exists globally; many are readily cross-referenced to clinical records. A wealth of information is thus potentially available to search for biomarkers among expressed genes using whole-transcriptome profiling. However, RNA from FFPE samples is generally severely degraded and, in the absence of enrichment methods, rRNA reads account for a large portion of the deep sequencing information.
Epicentre's Ribo-Zero™ kits provide a reliable method to deplete rRNA from FFPE samples, enabling whole-transcriptome RNA-Seq. A recent report by Morlan et al. describes another method, called Selective Depletion of abundant RNA Species (SDRNA): the use of DNA probes homologous to human cytoplasmic and mitochondrial rRNAs. RNA was purified using the MasterPure™ RNA Purification Kit, treated with either SDRNA or poly(A) enrichment, libraries were prepared using the Illumina® mRNA-Seq Kit or Epicentre's ScriptSeq™ mRNA-Seq Kit (now replaced by the ScriptSeq v2 Kit), and sequenced on the GAII and HiSeq 2000 instruments.
Libraries from SDRNA exhibited <1% rRNA content in uniquely mapped reads. When SDRNA libraries were compared to a poly(A)+ library, the outlier sequences were primarily identified as snRNAs, snoRNAs, scRNAs, and mirRNAs, demonstrating that a SDRNA library better represents the whole transcriptome than a poly(A)+ library. The authors mention that the Ribo-Zero Gold Kit is the only commercially available method that can effectively deplete rRNA from fragmented total RNA, and produced very comparable results to the method described in the article "with respect to the magnitude of depletion and hands-on time."
Morlan JD et al. (2012). Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue. PloS one, 7 (8) PMID: 22900061
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Morlan JD et al. (2012) Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue. PloS one, 7(8). PMID: 22900061
Rapid Amplification of cDNA Ends (RACE), the method of choice to study RNA transcription start sites, can be readily accomplished using enzymatic processing of RNA for downstream PCR. Kim et al. report a genome-wide survey of transcription start sites (TSS) in the pathogenic bacteria Escherichia coli and Klebsiella pneumoniae.
The method used two unique Epicentre enzymes, RNA 5'-Polyphosphatase and Terminator Exonuclease, as part of a procedure to isolate and process mRNA from bacterial total RNA. The survey generated important information by locating transcription starts, 5'-untranslated regions, and other regulatory regions found in the transcriptome, enabling a comparison of two otherwise closely related organisms.
The technique outlined in the report is similar to that in the ExactSTART™ 5' & 3' RACE Kit. Total RNA was treated with Terminator Exonuclease to remove rRNA and degraded mRNA, enriching the sample in full-length mRNA containing 5'-triphosphorylated ends. The mRNA was treated with 5'-RNA Polyphosphatase to convert the 5'-triphosphates into 5'-monophosphates. The 5'-monophosphorylated mRNA was ligated to an adaptor molecule containing Illumina sequences using T4 RNA Ligase, and then reverse-transcribed using a modified Illumina RT primer. The resulting library was PCR-amplified and sequenced on an Illumina GAII sequencer.
The data generated were used to perform comparative genomic/gene expression studies between the two closely related organisms; the researchers identified 3,746 and 3,143 TSSs for E. coli and K. pneumoniae, respectively. Comparison of promoter regions and other regulatory sites revealed that 70% of the primary transcripts were expressed during exponential growth; this similarity changed dramatically when comparing TSS and regulatory elements.
The authors conclude that their comparative approach provides "a starting point for the determination of conserved and specific features of the transcriptional output of closely related bacteria at single nucleotide resolution."
Kim D et al. (2012). Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling. PLoS Genetics, 8 (8) PMID: 22912590
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Kim D et al. (2012) Comparative Analysis of Regulatory Elements between Escherichia coli and Klebsiella pneumoniae by Genome-Wide Transcription Start Site Profiling. PLoS Genetics, 8(8). PMID: 22912590
The EZ-Tn5™ Transposome System is now finding favor in studies of more unusual environmental microbial species. The bacterial phylum Planctomycetes is found in various environmental habitats, as well as in the gastrointestinal tract of many mammals and fish. Members of this phylum possess characteristics that make them, at first glance, unsuitable for in vivo transposition experiments to develop a model genetic system. Although genome sequencing data are available for several Planctomycetes species, the lack of molecular genetic tools has proved challenging to more in-depth study of these organisms.
Schrier et al. describe the use of the EZ-Tn5 R6Kγori/KAN-2 Transposome, co-electroporated into P. limnophilus with Epicentre's TypeOne™ Restriction Inhibitor, and successfully generated mutants in two separate experiments. Screening on kanamycin allowed the selection of transposon mutants, which were rescued using the R6Kγ origin of replication in the transposome. Recovered mutants were screened using PCR to identify those that contained an insertion within the pckA gene. The PckA gene product catalyzes the first step in the gluconeogenesis pathway; thus, a mutation in the gene's open-reading frame should result in growth inhibition in a medium not supplemented with glucose. This blockage in the mutant studied was found to be reversible, as addition of pyruvate appeared to restore wild-type growth in the absence of glucose.
The syudy is another example of the utility of the EZ-Tn5 Transposome system in unusual bacteria, allowing the generation and screening of mutants in specific genes to study behavior predicted from sequencing analysis.
Schreier HJ et al. (2012). Transposon Mutagenesis of Planctomyces limnophilus and Analysis of a pckA Mutant. Appl Environ Microbiol PMID: 22798371
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Schreier HJ et al. (2012) Transposon Mutagenesis of Planctomyces limnophilus and Analysis of a pckA Mutant. Appl Environ Microbiol. PMID: 22798371
Ritlop et al. describe the use of a custom-engineered EZ-Tn5™ transposon and a powerful genetic screen to investigate the autocatalytic splicing of group II introns from pre-mRNA. The self-splicing 3.5-kb L1.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis was the subject of the study.
The custom transposon contains a pepN transcriptional promoter followed by the P23 L. lactis constitutive promoter. In vitro transposition into a cloned gene created a library of clones with the transposon at every possible position. The genetic screen allowed growth of clones only if the transposon had inserted into the group II intron such that the transcribed RNA fragments were able to assemble, trans-splice, and accurately ligate the two flanking exons. Of 201 independent clones sequenced, the Tn5 transposon was exclusively found in the sense orientation with respect to the intron, such that the terminator and promoter were functional and the intron was transcribed in two fragments. All 201 clones were unique. No clones were identified with the transposon in functionally important regions of the intron.
The study demonstrated the diverse utility of Tn5-based transposons. Further, the discovery that group II introns may be fragmented into two or three pieces and still self-assemble and splice properly provides support for an evolutionary relationship between group II introns and spliceosomal small nuclear RNAs.
Ritlop, C. et al. (2012). Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen PLoS ONE, 7 (8) DOI: 10.1371/journal.pone.0041589... Read more »
Ritlop, C. et al. (2012) Isolation and Characterization of Functional Tripartite Group II Introns Using a Tn5-Based Genetic Screen. PLoS ONE, 7(8). DOI: 10.1371/journal.pone.0041589
Stadler et al. investigated the molecular effects of miRNA regulation on certain well-characterized C. elegans genes using high-throughput ribosome profiling, a novel method to study translation of mRNAs and translation control by miRNAs. The method uses Epicentre's Circligase™ enzyme to generate sequencing template (see Figures 1 and 5 in Epicentre's poster: 1.8 MB PDF).
Briefly, ribosomes were isolated and concentrated by sucrose-gradient centrifugation, and treated with a ribonuclease to remove extraneous RNA from the ribosome preparation. Next, the poly(A) portion of the RNA was isolated from the ribosomal proteins, followed by size selection. Reverse transcription of the enriched poly(A) mRNA was performed using a novel primer that contained Illumina® sequences P5 and P7 for bridge PCR/cluster generation on a flow cell and sequencing priming sites, as well as an abasic site that allows for chemical cleavage. Circligase enzyme was used to recircularize the size-selected, single-stranded (ss) DNA. Following recircularization, the ssDNA templates were relinearized using chemical methods and PCR-enriched. The templates were then sequenced on an Illumina GAII instrument.
In this study, the authors were able to compare the relative abundance of the footprinted RNA with total mRNA isolated from the cells; they determined that functional down-regulation by some miRNAs was associated with decreases in both overall mRNA abundance and ribosome loading. They also determined that the changes were of substantially smaller magnitude than corresponding changes observed in translated protein abundance. Other miRNA targets showed only modest effects, consistent with models in which miRNA-mediated regulation occurs through a combination of mechanisms.
Stadler M et al. (2012). Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets. Genome Res. PMID: 22855835... Read more »
Stadler M et al. (2012) Contributions of mRNA abundance, ribosome loading, and post- or peri-translational effects to temporal repression of C. elegans heterochronic miRNA targets. Genome Res. PMID: 22855835
Many FFPE tissue blocks containing samples related to known disease states are archived in a multitude of hospitals worldwide. The RNA available from these blocks is not only limited in quantity but also generally severely degraded. In this report, Sinicropi et. al., discuss methods for RNA-Seq using RNA isolated from archival FFPE samples with low input levels of RNA, and compare the results to data previously generated by RT-PCR analysis.
The authors used the MasterPure™ RNA Purification Kit to isolate total RNA from 136 FFPE primary breast cancer tumor specimens. The isolated RNA was used as input for the ScriptSeq™ mRNA-Seq Library Preparation Kit (now replaced by the ScriptSeq v2 Kit) for preparing whole-transcriptome libraries. Sequencing was performed on an Illumina HiSeq 2000 instrument. Analysis showed that the RNA-Seq data generated using 5- to 12-year-old FFPE tumor tissue compared very favorably with RT-PCR results published earlier, and enabled quantification of transcripts with accuracy and sensitivity sufficient for biomarker discovery. The study identified >1,300 transcripts associated with breast cancer risk; more than half of the RNAs identified as prognostic were noncoding. Thus, the study demonstrated the value of a whole-transcriptome approach, and the results obtained should prove valuable in future screens of biomarkers associated with breast cancer recurrence risk.
Sinicropi D et al. (2012). Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue. PloS one, 7 (7) PMID: 22808097... Read more »
Sinicropi D et al. (2012) Whole Transcriptome RNA-Seq Analysis of Breast Cancer Recurrence Risk Using Formalin-Fixed Paraffin-Embedded Tumor Tissue. PloS one, 7(7). PMID: 22808097
Multiple sclerosis (MS) is a devastating illness with no known cause or cure. Recently, Laska et al. revealed that onset of the illness appears to increase expression of a specific retrovirus, HERV-Fc1, that belongs to the HERV-H/F family.
The study included investigating expression of certain components of HERV-Fc1 by qRT-PCR. During the studies, standards for various expressed transcripts of the virus were generated using the Durascribe® T7 Transcription Kit. The Durascribe Kit synthesizes RNA that contains 2'-fluorouracil and 2'-fluorocytidine, rendering transcripts resistant to degradation by ribonucleases A, B, and C. The standards were used as controls to determine the extracellular HERV-Fc1 RNA load in normal and MS individuals. The analysis showed that the expression of a capsid (gag) protein of HERV-H/F was significantly increased in CD4+ (P <0.001) and CD8+ (P <0.001) T lymphocytes, and in monocytes (P = 0.0356) from MS patients with active disease.
The importance of T cells in MS induction and further progression has been well documented. The methods developed in this study will enable further investigation of differential expression of endogenous retroviral markers in HERV-Fc1 biology related to MS, and possibly other autoimmune disorders.
Laska MJ et al (2012). Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active multiple sclerosis. Journal of virology, 86 (7), 3713-22 PMID: 22278236... Read more »
Laska MJ et al. (2012) Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active multiple sclerosis. Journal of virology, 86(7), 3713-22. PMID: 22278236
EZ-Tn5™ Transposomes are popular for generation of insertion and knockout mutants in microorganisms, 12 years after their introduction. A recent report by Veeranagouda et al. describes the generation of mutants in the anaerobe Bacteroides fragilis. The study is notable since anaerobes tend to be quite difficult to mutagenize through transposition, due to electroporation in the presence of oxygen that can poison the organism.
The experiments performed on B. fragilis used a custom transposome generated using the pMOD-3 [R6Kγ/MCS] vector. Screening/antibiotic resistance markers for erythromycin and kanamycin were introduced into the vector. As Bacteroides has a restriction system that requires bypassing, the pMOD3 construct (referred to as pVY02) was passaged through a strain of B. fragilis to methylate and protect the plasmid containing the transposon construct in a process called "transposon laundering" (Barry Hall, University of Rochester, personal communication). The transposon was released and purified from pYV02 using restriction digestion with Pvu II, gel-purified, and used to prepare the transposome complex with EZ-Tn5 Transposase. The transposome was electroporated into B. fragilis strain BF638 using standard methods and transposition mutants were recovered by screening on Brain/Heart Infusion (BHI) agar/erythromycin plates.
Results showed the transposon inserted in vivo at a relatively high frequency into BF638R, yielding 3.2 ± 0.35 x 10^3 CFU/µg. Transposome laundering resulted in a 6-fold increase in the number of transposome mutants over the nonprotected transposome controls. The use of the EZ-Tn5 pMOD-3 vector in the transposome construction process facilitated marker rescue by restriction digestion of DNA from the transposed host, recircularization, and transformation of the rescued DNA in E. coli strain EC100D (pir+). The transposome inserted into the Bacteroides genome in a single copy, demonstrated by Southern blotting and by single random-primer PCR (similar to the Random Amplification of Transposome Ends method). Further work by the researchers also demonstrated that the custom transposome was also useful in the mutagenesis of the bacterium Bacteroides thetaiotaomicron.
Veeranagouda, Y et al. (2012). Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome. FEMS Microbiology Letters PMID: 22639975... Read more »
Veeranagouda, Y et al. (2012) Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome. FEMS Microbiology Letters. PMID: 22639975
Genomic analysis has greatly increased our understanding of bacterial-insect interactions. Yet there remains a need for effective methods to remove insect rRNA to enable comprehensive, transcriptome-based studies. In a recent publication, Kumar et al. investigated the performance of the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) with insect samples. Eggs derived from a horizontal gene transfer of the Wolbachia bacterial genome into Drosophila ananassae were chosen as a model system. The authors performed paired-end RNA-Seq of untreated and Ribo-Zero-treated D. ananassae egg RNA using the Illumina® GAIIx system.
The authors conclude that the Ribo-Zero Kit (designed for human samples) efficiently removed >98% of D. ananassae rRNA from total RNA samples and that the Ribo-Zero-treated sample yielded a 6.2-fold increase in detection of mRNA transcripts. They further conclude that, based on their data, three times as many transcripts can be analyzed in a differential gene expression study that requires at least 100 reads per transcript. Finally, in D. ananassae, as in many insects, the 23S rRNA is naturally cleaved into two fragments. The Ribo-Zero treatment was found to efficiently remove both halves of the cleaved rRNA.
Kumar, N. et al. (2012). Efficient subtraction of insect rRNA prior to transcriptome analysis of
Wolbachia-Drosophila lateral gene transfer BMC Res Notes, 5 DOI: 10.1186/1756-0500-5-230... Read more »
Kumar, N. et al. (2012) Efficient subtraction of insect rRNA prior to transcriptome analysis of Wolbachia-Drosophila lateral gene transfer. BMC Res Notes, 230. DOI: 10.1186/1756-0500-5-230
The EZ-Tn5™ R6Kγori/KAN-2 Transposome has been used for elucidation of bacterial gene structure and function for the last 12 years. In a recent publication by Khatiwara et al., it was used to mutagenize a strain of Salmonella enterica serotype Typhimurium to link specific gene expression information to the gene of interest. The mutagenesis work also demonstrates a new method of transposome insertion sequencing using Illumina technology instead of older Sanger sequencing techniques.
The researchers noted that the 19-base mosaic end (ME) of the transposome contains a DNA sequence similar to the recognition sequence of type II restriction enzyme BsmF I (recognition site 5'-GGGAC(N)10|/3'-CCCTG(N)14|5'), except for one nucleotide. Since BsmF I cuts the site 14 bp away from the recognition site, the researchers exploited this finding to extract 12-nucleotide sequences immediately adjacent to Tn5 insertion sites. These 12-bp transposon-junction sequences were selectively amplified from a mutant library and sequenced. The resulting profile provides information on both the identity and relative quantity of each insertion in the library. Next, the researchers tested whether any mutagenesis in the ME would negatively affect the activity of the transposome. The results suggested that a single nucleotide change in the ME would not affect the transposition reaction significantly, as determined by comparative numbers of Kan-resistant colonies after mutagenesis.
One of the MEs was mutated from A to G to create the BsmF I site. This change allowed the ligation of a "Tn-Seq" linker to the mutated ME, which provided an Illumina single-read sequencing priming site and index into the molecule. The transposome generated was electroporated into the Salmonella spp. bacterium and allowed to generate colonies as per the normal in vivo transposition protocol. Mutants were then screened for changes in activity of selected loci. Individual mutants with expression changes in the same locus were pooled, digested with BsmF I, ligated to the Tn-Seq linkers and amplified by PCR, using a short extension time to generate short amplicons for single-read Illumina sequencing with five separate indices.
Sequencing information permitted mapping of the transposition mutants and then mapping the transposon insertions that provided changes in gene expression activity. The study highlights the adaptations researchers are developing to enable deep sequencing applications for gene expression studies in bacteria.
Khatiwara, A. et al. (2012). Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium Applied and Environmental Microbiology, 78 (9), 3098-3107 DOI: 10.1128/AEM.06865-11
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Khatiwara, A. et al. (2012) Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium. Applied and Environmental Microbiology, 78(9), 3098-3107. DOI: 10.1128/AEM.06865-11
Microbial geneticists continue to use the EZ-Tn5 in vivo transposomics tools for establishing genetic systems in novel bacteria. Jogler et al. report on the Planctomycetes phylum of bacteria which is a major component of the global nitrogen and carbon cycles. These bacteria perform reactions such as the anaerobic oxidation of ammonium ions. Many Planctomycetes strains are difficult to cultivate, so the researchers developed a model system using the P. limnophilus strain due to its cultivability, sensitivity to kanamycin, rapid growth, and availability of the genome sequence.
The authors used the EZ-Tn5 <R6Kgamma/KAN-2> Transposome in a standard in vivo transposition reaction to generate mutants that were detected by i) Kan resistance and ii) modified arbitrary PCR. The mutants were screened using a second arbitrary PCR step. Analysis showed that nine mutations were generated that were pinpointed to specific regions on the chromosome. The authors comment that there appeared to be only insignificant regional bias in the insertion sites. The authors further noted that the intracellular membrane and the condensed nucleoid did not appear to have any negative effect on the transposition; thus, the likelihood of gene transfer and mutagenesis in Planctobacillus spp. appears to be quite good. These results will enable future in-depth genetic analysis of Planctomycetes.
Jogler, C. et al. (2011). Characterization of Planctomyces limnophilus and Development of Genetic Tools for Its Manipulation Establish It as a Model Species for the Phylum Planctomycetes Applied and Environmental Microbiology, 77 (16), 5826-5829 DOI: 10.1128/AEM.05132-11... Read more »
Jogler, C. et al. (2011) Characterization of Planctomyces limnophilus and Development of Genetic Tools for Its Manipulation Establish It as a Model Species for the Phylum Planctomycetes. Applied and Environmental Microbiology, 77(16), 5826-5829. DOI: 10.1128/AEM.05132-11
The Tasmanian devil was recently listed as an endangered species, primarily due to the emergence of a fatal, transmissible cancer known as devil facial tumour disease (DFTD). The disease was recently reported in Northern Tasmania, and has resulted in depletion of populations that could ultimately make the animals extinct within 25 to 35 years. Recently, Deakin et al. conducted a genetic survey of a BAC library created with the CopyControl™ BAC Library Construction Kit (EcoR I) from genomic DNA extracted from the liver of a deceased two-year-old male Tasmanian devil. Physical maps were compared to the genomes of other marsupials to determine the genesis of this particular disease.
The results showed that massive genomic restructuring appears to be a catalyst for development of the DFTD syndrome, based on chromosome painting and fluorescence in situ hybridization (FISH) experiments conducted using the BAC library. The authors developed a detailed map of the global chromosome restructuring and intricate gene rearrangements that characterize DFTD. Only limited regions of the genome were found to be highly rearranged. After the rearrangements occur, the tumor karyotype is remarkably stable during its clonal transmission from animal to animal. By anchoring genes to a reference and tumor maps, the authors believe that they can predict the locations of common tumor suppressor genes and oncogenes. This study provides an important framework for future genomic studies into DFTD and enhances the value of the creation of large-insert genomic libraries.
Deakin, J. et al. (2012). Genomic Restructuring in the Tasmanian Devil Facial Tumour: Chromosome Painting and Gene Mapping Provide Clues to Evolution of a Transmissible Tumour PLoS Genetics, 8 (2) DOI: 10.1371/journal.pgen.1002483
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Deakin, J. et al. (2012) Genomic Restructuring in the Tasmanian Devil Facial Tumour: Chromosome Painting and Gene Mapping Provide Clues to Evolution of a Transmissible Tumour. PLoS Genetics, 8(2). DOI: 10.1371/journal.pgen.1002483
The role of DNA methylation in gene regulation and epigenetics is of ever-increasing interest. Until now, investigating DNA methylation has been crippled either by: i) the necessity for microgram quantities of input DNA for whole-genome bisulfite sequencing (WGBS); or ii) incomplete interrogation of the methylome in the case of reduced-representation bisulfite sequencing (RRBS).
In a recent publication, Adey and Shendure report a transposon-based in vitro library construction method to demonstrate that a highly complex bisulfite sequencing library may be obtained from as little as 10 ng of genomic DNA. The authors describe the use of a custom-loaded transposase for "tagmentation" (tagging and fragmentation of the DNA) that contained an unmethylated transposon sequence. After an oligonucleotide replacement procedure, in which (methylated) adaptors were ligated to each end of the tagmented genomic DNA, standard bisulfite treatment was used. The library was amplified by PCR and sequenced on an Illumina® MiSeq using a single-end, 100-bp run. Over 100 million aligned reads were obtained at high complexity with >96% of CpG and >98% of non-CpG cytosines covered for the genome. The necessity to have part of the transposon DNA unmethylated served as an internal control and demonstrated that the conversion rate of the bisulfite treatment was >99%. The method described will allow for whole-methylome interrogation where DNA sample is limiting (biopsy samples, microdissected tissues, etc.) and may also allow for the utilization of poor-quality or degraded DNA samples.
Adey, A., & Shendure, J. (2012). Ultra-low-input, tagmentation-based whole genome bisulfite sequencing Genome Research DOI: 10.1101/gr.136242.111... Read more »
Adey, A., & Shendure, J. (2012) Ultra-low-input, tagmentation-based whole genome bisulfite sequencing. Genome Research. DOI: 10.1101/gr.136242.111
Although much of RNA-Seq research has focused on the human transcriptome, there is considerable interest in applications of RNA-Seq to environmental metatranscriptomics samples. Giannoukos et al. at the Broad Institute evaluated several methods for removal of ribosomal RNA (rRNA) from total RNA preparations of complex microbial communities in order to maximize RNA-Seq reads. They also examined bacterial populations from clinical human stool samples.
The researchers used total RNA from well-characterized microbial culture samples individually, as well as a mixed pool, for benchmarking rRNA removal methods:
Epicentre Ribo-Zero™ Meta-Bacteria Kit (solution-based hybridization capture);
NuGEN Ovation Prokaryotic RNA-Seq System (NuGEN) (proprietary set of "not so random" primers to avoid rRNA as template during first and second strand cDNA synthesis);
Duplex-Specific Nuclease (DSN; degradation of fast re-annealing abundant cDNAs to preferentially deplete ribosomal cDNA);
Life Technologies MicrobEXPRESS Kit (solution-based hybridization capture); and
Epicentre Terminator Exonuclease (degrades RNAs with a 5'-monophosphate group).
The authors constructed RNA-Seq libraries after using each of the methods above, sequenced them on the Illumina platform, and mapped the reads to the corresponding reference genomes. For the clinical samples, they mapped reads to 649 bacterial reference genomes.
The results demonstrated the superiority of the Ribo-Zero Kit in reducing rRNA in the microbial samples. In artificially degraded samples and in the clinical samples, the Ribo-Zero Kit performed well, while some of the other methods performed poorly or failed completely at removing rRNA. The authors further note the potential for simple automation of the Ribo-Zero procedure, and that the Ribo-Zero process can be applied to profile gene expression in both simple and complex, naturally occurring bacterial communities.
Giannoukos, G. et al. (2012). Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes Genome Biology, 13 (3) DOI: 10.1186/gb-2012-13-3-r23... Read more »
Giannoukos, G. et al. (2012) Efficient and robust RNA-seq process for cultured bacteria and complex community transcriptomes. Genome Biology, 13(3). DOI: 10.1186/gb-2012-13-3-r23
A recent study by Koerner et al. cites the use of Epicentre's most popular products: the Ribo-Zero™ Kit (Human/Mouse/Rat) to deplete rRNA, and the ScriptSeq™ Kit to prepare RNA-Seq libraries. The authors explored the presence of CpG islands at the 5' end of the Airn noncoding RNA (ncRNA). This region contains some unusual features, such as a GC-rich region just downstream from the promoter, and tandem direct repeats (TDR) in its second half.
The authors used homologous recombination to generate embryonic stem cells that carry deletions at the endogenous locus of the entire CpG island or only the TDRs. The TDRs were shown to play a small part in the Airn transcription elongation process, but a much larger part in methylation of the Airn promoter that comes from the maternal allele. Further, the CpG island is essential for transcription initiation of Airn, and in maintaining the unmethylated state of the Airn promoter. The research provides insight into how the imprinted Airn ncRNA CpG island (normally methylated on the maternal and unmethylated on the paternal chromosome) regulates its associated promoter, suggesting that a class of CpG islands can exhibit regulatory effects on upstream transcriptional elements.
Koerner, M. et al. (2012). A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter PLoS Genetics, 8 (3) DOI: 10.1371/journal.pgen.1002540
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Koerner, M. et al. (2012) A Downstream CpG Island Controls Transcript Initiation and Elongation and the Methylation State of the Imprinted Airn Macro ncRNA Promoter. PLoS Genetics, 8(3). DOI: 10.1371/journal.pgen.1002540
Transposon mutagenesis is still considered to be an excellent tool for probing the genomics of bacterial species to create gene-silencing mutations. This approach can help elucidate the genetic markers responsible for various bioactivities.
A report by Garavaglia et al. explores the pathway of biosynthetic genes responsible for biofilm formation in the bacterium E. coli MG1655. Using the EZ-Tn5™ <R6Kγori/KAN-2> Transposome, insertional mutants were characterized that allowed the researchers to determine that blocking transcription of the csgDEFG operon in the uridine monophosphate (UMP) biosynthesis pathway inhibits biofilm formation. The transposon mutation was found to be in the carB gene, encoding a subunit of carbamoyl phosphate synthetase, which catalyzes the first step in the de novo pyrimidine nucleotide biosynthetic pathway.
A requirement of biofilm formation is the production of cellulose by E. coli, and this biosynthesis is modulated by the UMP pathway. Mutations in the carB gene also shut down cellulose production, as shown by lack of biofilm formation. Adding back exogenous uracil (which can be converted to UMP through the pyrimidine nucleotide salvage pathway) restored cellulose production and biofilm formation. Thus, the authors conclude that there exist tight links between pyrimidine metabolism and cellulose production/biofilm formation.
Garavaglia, M. et al. (2012). The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031252
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Garavaglia, M. et al. (2012) The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli. PLoS ONE, 7(2). DOI: 10.1371/journal.pone.0031252
Göransson et al. report a new method of finding pathogens as part of a biosecurity study that is able to detect pathogens down to the single-molecule/organism level. The method combines a padlock probe approach, using Ampligase® Thermostable DNA Ligase, with rolling-circle amplification (RCA).
The authors used an environmental air sampling unit to trap particulate material on a membrane, followed by a rapid extraction of the DNA using magnetic beads. After clean-up, the DNA-containing solution was placed into an "on-bead" padlock probe/proximity ligation assay (PLA) catalyzed by Ampligase enzyme. Reacted probes were then subjected to two further rounds of RCA, first on beads and then in solution. Probes were then tagged with fluorescent dye and detected using an optical system with sensitivity down to 30 bacteria or 5 spores. The authors have improved the performance of the system by reducing the time required for the RCA step and are working improve the sensitivity of the process.
The combination of improved RCA and sensitive detection represent a significant improvement over previous methods for pathogen detection. The method can also be adapted to detect proteins.
Göransson, J. et al. (2012). Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0031068
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Göransson, J. et al. (2012) Rapid Identification of Bio-Molecules Applied for Detection of Biosecurity Agents Using Rolling Circle Amplification. PLoS ONE, 7(2). DOI: 10.1371/journal.pone.0031068
RNA-Seq techniques are well integrated into the study of cancer genetics and are useful in locating splice variants in genes that may be indicative of a malignant cell. In a recent study, Salzman et al. observed, using RNA-Seq analysis, that a large amount of spliced transcripts isolated from cancer cells are actually circular in nature. The results suggest that other splicing modes are possible and may be useful in differentiating cancerous from normal tissue types. So-called "scrambled exons", which are evident by splicing order errors in various mRNA species, are likely a result of the circularization of these transcripts.
To arrive at this conclusion, the authors initially isolated total RNA from bone marrow cells, removed rRNA using the Ribo-Zero™ Kit (Human/Mouse/Rat), prepared RNA-Seq libraries using the Illumina TruSeq RNA-Seq Sample Prep Kit, and sequenced the libraries on an Illumina GAIIx sequencer. Leukemia samples were sequenced together, while remission blood samples and normal bone marrow subpopulations were sequenced in separate runs. In another analysis, the authors used paired-end mapping ratios to determine the relative abundance of scrambled isoforms to normal linear isoforms. Each gene was tiled by dividing it into even-length bins of 200 bp. Data were validated by qRT-PCR.
The researchers further concluded that the scrambled splicing activity appears to occur most commonly in the cytoplasm. Four potential models for circularization were presented, using techniques that also demonstrated the presence of circular RNA splice variants using RNase R exoribonuclease.
Salzman, J. et al. (2012). Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types PLoS ONE, 7 (2) DOI: 10.1371/journal.pone.0030733
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Salzman, J. et al. (2012) Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types. PLoS ONE, 7(2). DOI: 10.1371/journal.pone.0030733
RNAse R is a unique Epicentre enzyme that is finding greater use in studying single-stranded RNAs, including circular RNAs that contain linear single protruding strands ("lariats"). These molecules have important biological functions, including roles in viral life cycles and tRNA maturation .However, discovery of circular RNAs has so far been mostly serendipitous, and methods to study these molecules are needed.
Danan et al. developed a directed method to pinpoint RNA-Seq reads that have a permuted mapping to the genome, a characteristic of circular RNA. They developed a workflow to enrich for circular transcripts and overcome possible artifacts, by pretreating the RNA sample using RNase R. The isolated circular RNA was used in the development of a new sequencing method, "circRNA-Seq", which uses enriched circular RNAs and allows quantification of relative abundance/prevalence of these RNAs in the cell in an unbiased way. The authors applied the technique to the archaeon Sulfolobus solfataricus P2. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but also many noncoding RNAs and circular RNAs of unknown function. Many of the identified circles were conserved in S. acidocaldarius, further supporting their functional significance. The data suggest that circular RNAs, especially circular noncoding RNAs, are more common in archaea than previously recognized. The circRNA-seq method will enable the study of these novel RNAs in any organism and will help to determine their relative importance in the biology of the cell.
Danan, M. et al. (2011). Transcriptome-wide discovery of circular RNAs in Archaea Nucleic Acids Research DOI: 10.1093/nar/gkr1009
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Danan, M. et al. (2011) Transcriptome-wide discovery of circular RNAs in Archaea. Nucleic Acids Research. DOI: 10.1093/nar/gkr1009
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