Thursday, December 23, 2010

Comparison of various DNA-Seq library prep methods

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 prepare literally hundreds of libraries in a day.  With respect to bias, the authors state: 
Comparison to conventional methods of library preparation, relying on mechanical or endonuclease fragmentation, finds that although transposase-catalyzed adaptor insertion demonstrates a slightly greater insertion bias, this has little impact at the level of genomic coverage, and is offset by large advantages with respect to speed, simplicity, and low input requirements. 
As described in the paper, the Nextera system is highly versatile, and can be adapted to multiple applications. These include library preparation from as little as 10 pg DNA, exome capture, PCR-free and colony PCR library preparation, and sample prep automation.

ResearchBlogging.orgAdey, 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

Monday, December 20, 2010

Quantitation of input DNA and Nextera™ fragment size distribution

When preparing Illumina-compatible Nextera libraries, it is critical to accurately quantify the input DNA amount. Variation in input DNA can affect the molecular weight (MW) distribution of libraries. The Bioanalyzer traces below show size distribution as a function of input DNA amount (human genomic DNA). As the traces show, the MW distribution increases (larger fragments) with the input DNA amount. Please note that the degree of variance will depend on a number of factors, including sample type and purity. For accurate DNA quantitation, any of the following methods can be used: qPCR, Qubit® fluorimetry, or NanoDrop™ analysis.

If a stringent size distribution is needed, there are a number of methods that can be used to size-select for the required fragment size, including AMPure® XP (for removing small fragments), Caliper LabChip® XT (for very narrow size selection), or gel extraction.


Fragment size and amount of input DNA (click to enlarge figure). Varying amounts of human genomic DNA were tagmented with Illumina-compatible Nextera™ Enzyme Mix. Tagmentation was performed using the HMW buffer, followed by nine cycles of PCR and DNA clean-up (without size selection). Red: 25 ng; Blue: 50 ng; Green: 100 ng; Aqua: 150 ng.

Wednesday, December 15, 2010

BAC libraries enable phylogenetic analysis in Japanese pear

With the rapid expansion of next-generation sequencing, there has been speculation that the backbone technology of older genomic sequencing, large-insert cloning (e.g., using bacterial artificial chromosomes [BACs]) would become less important. However, in many laboratories, BAC libraries still play a role in sequencing and gene expression studies.

In a recent study, Okada et al. screened a library created in the CopyControl™ BAC Cloning Kit (Hind III cloning-ready vector) to study the mechanics of self-incompatibility (inhibiting self-fertilization) in the Japanese pear (Pyrus pyrifolia). The work focused on a DNA contig that contained a 649-kb region around the S-RNase genes. After creation of the BAC library, the selected clones were sequenced using standard ABI BigDye 3.1 sequencing techniques and “old-fashioned” chromosome walking. However, BAC library screening services are increasing turning to next-generation sequencing technologies, due to the ease of rapidly generating large amounts of sequence data with better depth and coverage than traditional Sanger sequencing.

ResearchBlogging.orgOkada, K. et al. (2010). Related polymorphic F-box protein genes between haplotypes clustering in the BAC contig sequences around the S-RNase of Japanese pear Journal of Experimental Botany DOI: 10.1093/jxb/erq381

Friday, December 10, 2010

Microsatellite loci for Symbiodinium A3 Identified using next-generation sequencing

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-algal symbioses. For this study, genomic DNA from three cultured strains of S. fitti were extracted and purified. Two different methods were employed to identify microsatellite loci with di-, tri-, and tetranucleotide motifs: i) Roche 454 sequencing; and ii) standard clone library (TA cloning) amplified and cycle sequenced using the ABI’s Big Dye Terminator Kit. For the Roche 454 sequencing, libraries were prepared from only 50 ng of double-stranded DNA using the Nextera™ DNA Sample Prep Kit (FLX Titanium-compatible), and sequenced on the 454 GS-FLX sequencer. The sequencing results helped the authors identify three to eight alleles for each haploid locus, with <95% of the samples possessing a single, symbiont, multilocus genotype (MLG).

The study demonstrates the utility of next-generation sequencing (NGS), especially with limited amounts of DNA. As shown in this study, NGS can be used to identify population genetic markers, which can help scientists better understand intraspecific and interspecific gene flow and population genetic structure.

ResearchBlogging.orgPinzón, 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

Thursday, December 2, 2010

New small-RNA-Seq library prep kit

Epicentre recently introduced the ScriptMiner™ Small RNA-Seq Library Preparation Kit (SinglePlex; Illumina-compatible). The kit produces nonbarcoded (singleplex) sequencing libraries from miRNA and, optionally, small 5'-capped RNA and 5'-triphosphorylated RNA. The ScriptMiner process includes a unique enzymatic procedure that removes excess 3' adaptor oligo, greatly reducing the amount of adaptor-dimer products in the sequencing library.


Small-RNA coverage from ScriptMiner™ and conventional libraries (click to enlarge figure). Small-RNA libraries were prepared using ScriptMiner (left panel) and conventional (right panel) methods and sequenced on an Illumina® GAIIx sequencer.  Both libraries produced similar numbers of aligned reads; however, the ScriptMiner library had a higher proportion of mature miRNA sequences.

Friday, November 12, 2010

New kits for rRNA removal

Epicentre’s Ribo-Zero™ rRNA Removal Kits are gaining wide acceptance as the new standard for removing rRNA from intact and partially degraded RNA samples for RNA-Seq library preparation. Two new Ribo-Zero Kits are now available:

The Ribo-Zero rRNA Removal Kit (Gram-Positive Bacteria) removes >99% of 23S and 16S rRNA and >93% of 5S rRNA from total RNA isolated from Gram-positive bacteria.

The Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) Low Input is ideal for removing rRNA from limited quantities of total RNA and from formalin-fixed paraffin-embedded (FFPE) RNA samples.

Wednesday, October 27, 2010

rRNA removal from Drosophila total RNA

We have received many inquiries from customers regarding the use of  the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) with RNA from nonmammalian organisms. Although we have not tested this kit with a wide range of genera and species, it will be suitable for many organisms whose rDNA genes exhibit a high degree of homology to human rDNA. However, the rRNA removal efficiency will vary, depending on the degree of homology.

One of our customers, Dr. Dominik Handler at the Institute of Molecular Biotechnology GmbH, Austria, examined the use of the Ribo-Zero (H/M/R) Kit with Drosophila RNA. He compared the amount of rRNA depletion with the Ribo-Zero kit and a competitive kit by qPCR after one and two rounds of rRNA removal.


qPCR analysis of rRNA depletion (click to enlarge figure). Total RNA samples were treated  with one or two rounds of the Ribo-Zero kit (RZ) or a competitive kit (R-), and fold depletion calculated using qPCR with primers to multiple regions of the indicated rRNAs, after normalization to rp49 mRNA. Nitric oxide synthase (nos) mRNA was used as an internal control.

In Drosophila, as in many diptera, the 28S rRNA is processed into two parts (a “left” and “right” arm). After two rounds of rRNA removal, Dr. Handler observed depletion of the left arm by 31,000X and the right arm by 600X. Overall, the Ribo-Zero kit performed substantially better than the competitive kit.


Wednesday, October 13, 2010

Obtaining optimal RNA-Seq library quality

The new ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina-compatible) prepares directional, ligation-free RNA-Seq libraries in less than 3 hours, from rRNA-depleted or poly(A)+ RNA. We compared the quality of ScriptSeq libraries prepared using different methods to treat the total RNA.

We used Universal Human Reference RNA (UHRR), Brain Reference RNA (BrRR), and total RNA isolated from FFPE breast cancer tissue as starting material. The specified samples were treated with either the Ribo-Zero™ Kit, a competitive rRNA-removal kit (Company A), or a commercial oligo(dT)-based mRNA enrichment kit. For UHRR and BrRR, ScriptSeq libraries were prepared from 50-ng aliquots of the resulting rRNA-depleted or poly(A)-enriched RNA. For FFPE samples, the entire amount of rRNA-depleted RNA recovered from 500 ng total RNA input was used to prepare the libraries. The di-tagged cDNA reactions were amplified by PCR for either 10 cycles (UHRR and BrRR) or 12 cycles (FFPE) followed by Exo I digestion. Each RNASeq library was purified using MinElute (Qiagen) and recovered in 15 μl of Elution Buffer. Replicate reactions were pooled and examined using a Bioanalyzer (Agilent). Single-lane, 54-nt unidirectional sequencing reads were obtained for each library using an Illumina GAII sequencer, and sequence analysis was performed using Bowtie.


Summary of sequencing data from ScriptSeq™ libraries (click to enlarge figure). Libraries were prepared as described above from Universal Human Reference RNA (UHRR), Brain Reference RNA (BrRR), or RNA extracted from FFPE breast cancer tissue (FFPE). The indicated method of rRNA removal or mRNA enrichment was used.

As seen in the figure, optimal RNA-Seq results were obtained with the Ribo-Zero Kit for rRNA removal, even from substantially degraded RNA (FFPE sample).

Thursday, October 7, 2010

Examining blocking lesions in ancient DNA

The characteristics of ancient DNA remain poorly understood. This is particularly true for blocking lesions (chemical alterations that cannot be bypassed by DNA polymerases). Blocking lesions prevent amplification and sequencing of affected molecules, thus limiting the analysis of DNA derived from ancient samples. Heyn et al. recently developed a new method--polymerase extension profiling (PEP)--that reveals occurrences of polymerase stalling on DNA templates. This sequencing-based technology allows detection of damage on a single-molecule level. The technique used CircLigase™ ssDNA Ligase for high-efficiency ligation of single-stranded adaptors (containing the Roche 454 A sequence) to the 3’ ends of primer-extension products.

The authors found evidence of blocking lesions in three out of four ancient samples, but no more than 40% of the molecules were affected, indicating that such modifications are far less frequent than previously thought.

ResearchBlogging.orgHeyn, P. et al. (2010). Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA Nucleic Acids Research, 38 (16) DOI: 10.1093/nar/gkq572

Thursday, September 30, 2010

Nextera library preparation technology used to characterize HIV intra-host diversity

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, followed by pyrosequencing. An average of 41,826 sequence reads per SIV genome was obtained, with an average coverage depth of 380 sequences. For HIV samples, an average of 29,000 sequence reads per genome with a sequencing depth range of 208-846 was obtained. Full or near-full coverage was obtained with Nextera libraries prepared from 50 ng of DNA.

The authors conclude that, using Nextera technology, they are able to demonstrate:
“...a new and highly practical approach to study the complexity of the viral population within a host and identify minor variants on a genome-wide scale. While this manuscript applies pyrosequencing to immunodeficiency viruses, this approach could be applied to any viral pathogen.”

ResearchBlogging.orgBimber, 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

Friday, September 24, 2010

Share your research results with EpiCentral readers!

If Epicentre products have helped you in your research, we’d like to hear about it. We invite you to write a guest post for our blog, and share your data with us and our readers.
  1. Posts should be approximately 500 words, with up to two figures or tables.
  2. Text may be submitted as Microsoft Word (.doc or .docx), RTF, or plain-text files. Images should be submitted as JPEG or TIFF files.
  3. Submit all materials by e-mail to: forum@epibio.com
  4. Upon publication, you will receive an honorarium of $100 credit towards the purchase of any Epicentre products, as well as a special Epicentre gift package valued at over $25.

Friday, September 17, 2010

Nextera libraries from buccal-cell DNA

We’ve received several inquiries regarding the use of Nextera™ technology to prepare next-generation sequencing libraries from buccal-cell DNA. The BuccalAmp™ DNA Extraction Kit is designed for a quick extraction of DNA for PCR only. However, our R&D scientists have developed a protocol, outlined below, to ensure successful preparation of Nextera libraries from DNA extracted with the BuccalAmp Kit. For this example, DNA was extracted from four buccal swabs and pooled.
  1. Centrifuge the tube briefly (1 minute at 3,000 x g) to remove the solid cellular debris.
  2. Remove the supernatant and precipitate the DNA with 1/10 volume of sodium acetate (3.0 M) and 2 volumes of ethanol.
  3. Resuspend the pellet in 100 µl of T10E1 buffer. Centrifuge briefly to remove insoluble material and transfer the supernatant to a DNA Clean & Concentrator-5 column (Zymo).
  4. Add 300 µl of Binding Buffer and follow the manufacturer’s protocol.
  5. Elute the DNA with two aliquots (10 µl each) of T10E1 buffer. Use 50 ng of eluted DNA, as determined spectrophotometrically, in the standard Nextera protocol.

Human buccal DNA after tagmentation reactions with the Nextera™ DNA Sample Prep Kit (Illumina-Compatible).  DNA was extracted and cleaned up as described above, and treated as follows: lane 1, no treatment ; lane 2, Hind III digestion; lane 3, Nextera tagmentation with HMW buffer; lane 4, Nextera tagmentation with LMW buffer. Lane M, 100-bp DNA ladder.

Thursday, September 9, 2010

Size distribution of Nextera Illumina-compatible libraries

Many of our Nextera™ customers have requested the ability to prepare libraries containing different sizes of DNA fragments for Illumina GAII sequencing. The Nextera™ DNA Sample Prep Kit (Illumina-compatible) now contains two buffers, Low-Molecular-Weight Buffer (LMW) and High-Molecular-Weight Buffer (HMW). The size distribution of the tagmented DNA can be controlled based on the buffer used in the reaction. Below are two Bioanalyzer traces from standard Nextera reactions using the indicated buffer.
LMW Buffer

HMW Buffer

We have also further optimized the reaction conditions to yield a narrow size distribution. For additional information on reaction conditions and on bias, library complexity, coverage, etc., please contact our technical support staff: techhelp@epibio.com

Thursday, September 2, 2010

HPV prevalence in esophageal squamous cell carcinoma

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 Australia were tested for the presence of HPV DNA by PCR, and positive samples were sequenced to determine HPV type. DNA was extracted from FFPE tissue blocks or slides using the QuickExtract™ FFPE DNA Extraction Kit. Samples were analyzed for the presence of HPV with general mucosal HPV primers, and β-globin PCR primers were used as a control. Of the 222 ESCC patients, only eight tested positive for HPV (six cases of HPV-16; two cases of HPV-35). None of 55 esophageal tissue controls from healthy patients tested positive for HPV. Lifestyle factors were also investigated in this study. Overall, there was weak evidence that that patients with HPV-positive ESCC had higher BMI than patients with HPV-negative tumors. The authors conclude that larger studies or pooled analyses will be required for definitive evidence regarding the role of HPV in ESCC.

ResearchBlogging.orgAntonsson, 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

Thursday, August 26, 2010

Defining the link between enterotoxin production and sporulation in C. perfringens

The second most common cause of bacterial foodborne illness is Clostridium perfringens type A. These isolates produce an enterotoxin (CPE), and an estimated 250,000 cases of resultant food poisoning occur annually in the U.S. Forty years ago, it was postulated that sporulation and enterotoxin production were linked and, in fact, C. perfringens type A isolates only produce CPE during sporulation.

Four sigma factors mediate sporulation in C. perfringens; however, the exact roles of two of them (SigF and SigG) are unknown. After confirming that sporulating wild-type SM101 cultures produce SigF and SigG, Li and McClane prepared isogenic sigF or sigG null mutants. They used the MasterPure™ Gram-Positive DNA Purification Kit to isolate DNA for Southern blotting, to confirm the presence of a single intron insertion in the SM101::sigF and SM101::sigG mutants. The detection of alternative sigma factor production by Western blot analysis was aided by Ready-Lyse™ Lysozyme. The authors concluded that all four sigma factors are needed for sporulation, but only SigE, SigF, and SigK are needed for synthesis of CPE. The results of this study indicate a previously unappreciated level of complexity for the regulation of cpe transcription.

ResearchBlogging.orgLi, J. and McClane, B. (2010). Evaluating the Involvement of Alternative Sigma Factors SigF and SigG in Clostridium perfringens Sporulation and Enterotoxin Synthesis Infect. Immun. : 10.1128/IAI.00528-10

Thursday, August 19, 2010

Using Nextera technology for RNA-Seq expression analysis


We’ve had several inquiries about using Nextera™ technology with cDNA, in order to generate libraries for RNA-Seq and subsequent gene expression analysis. We are grateful to the Richard M. Myers Laboratory at the Hudson Alpha Institute for Biotechnology (Huntsville, AL), for testing this application of Nextera technology. They used the following strategy (click on all figures to enlarge them):

Strategy for library preparation

With this strategy, decreased coverage at the ends of cDNAs is expected because two transposome insertions are required to produce a functional sequencing template.

Libraries using human RNA (ECC-1) were prepared with both the Nextera workflow and a standard RNA-Seq workflow, and sequenced on the Illumina GAII platform. Two Nextera libraries were prepared: one from 50 ng of cDNA, and the other from only 10 ng of cDNA. The results from both the conventional RNA-Seq and Nextera procedures compare very well:

Relative gene expression analysis


The data show that Nextera technology yields representative libraries, even with very low amounts of cDNA:

Correlation based on sample input amount


As seen from the coverage of the cDNA termini and library complexity, both libraries were highly complex and representative of the transcripts. The red arrows show the expected drop in coverage of 5' and 3’ ends with the Nextera library:

Library coverage and complexity

Monday, August 16, 2010

Purification of DNA and RNA from the same sample using the MasterPure Complete Kit

Many customers have asked how they can process both DNA and RNA from the same sample, using the MasterPure™ Complete DNA and RNA Purification Kit. The following protocol has been validated, and successfully used by customers:
  1. Follow the protocol for total nucleic acids (TNA) in the product literature, including Lysis (Part A) and the first two steps for Precipitation (Part B, steps 1 and 2).
At this point, split the supernatant into two equal portions. Label one tube “DNA” and the other “RNA”.
  1. Add 250 µl of isopropanol to both tubes, invert 30-40 times to mix.
  2. Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
  3. Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
  4. Rinse with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove residual ethanol with a pipet.
DNA Protocol (for the tube marked “DNA”)
  1. Resuspend the pellet in 100 µl TE Buffer.
  2. Add 1 µl of RNase A to sample and mix well.
  3. Incubate @ 37°C for 10 minutes. Note: Additional incubation (up to 30 minutes) may be needed.
  4. Add 100 µl of 2X T&C Lysis Solution, and mix by vortexing for 5 seconds.
  5. Add 100 µl of MPC protein precipitation reagent, mix by vortexing for 10 seconds. Place on ice for 3-5 minutes.
  6. Centrifuge for 10 minutes at ≥10,000 x g.
  7. Transfer the supernatant to a new microcentrifuge tube and discard the pellet.
  8. Add 250 µl of isopropanol. Invert 30-40 times to mix.
  9. Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
  10. Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
  11. Rinse twice with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove all residual ethanol with a pipet.
  12. Resuspend the DNA in 10-35 µl of TE Buffer.
RNA Protocol (for the tube marked “RNA”)
  1. Prepare 100 µl of DNase I solution by diluting 2.5 µl of RNase-Free DNase I up to 100 µl with 1X DNase buffer.
  2. Resuspend the pellet in 100 µl of DNase I solution.
  3. Continue with steps 3 through 12 from the DNA Protocol (above).

Wednesday, August 11, 2010

Ribo-Zero rRNA removal and gene expression analysis of fragmented RNA

A significant advantage of the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) is that it can remove virtually all the rRNA, even from degraded total RNA samples. We examined correlation of gene expression between RNA-Seq libraries prepared from intact and fragmented RNA samples treated with the Ribo-Zero Kit, as well as a competitive kit.

Intact and partially fragmented Universal Human Reference RNA (UHRR) samples (2 x 2.5 µg each) were treated with either the Ribo-Zero Kit or a competitive rRNA removal kit. The respective rRNA-depleted samples were pooled and, for each, RNA-Seq libraries were prepared in triplicate from the equivalent of 1 µg total RNA, using a random-primed cDNA synthesis method. Replicates of the respective RNA-Seq libraries were pooled and sequencing was performed using an Illumina GAIIx sequencer with 36-nt reads. The data were analyzed using Illumina’s Pipeline Eland_rna Module and CASAVA Software.

We observed a higher correlation (R2 = 0.9396) in genes detected between intact and fragmented Ribo-Zero rRNA-depleted RNA-Seq libraries (A) compared to the corresponding RNA-Seq libraries prepared using the competitive kit (R2 = 0.8940) (B). Also, an additional 1,016 genes were mapped (with ≥10 reads) for the Ribo-Zero RNA-Seq libraries, indicating better coverage of transcripts with reduced rRNA background.

A. Ribo-Zero rRNA-depleted RNA-Seq libraries


B. Competitor rRNA-depleted RNA-Seq libraries

Thursday, August 5, 2010

Fosmid cloning enables new techniques in synthetic biology

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 three chemical groups (alcohols, aldehydes, and organic acids). The authors located genetic functionalities on two fosmids that improved the growth of the E. coli host cell by 5.7- and 6.9-fold in the presence of these inhibitory compounds. They then produced chimeric clones that contained all of the desired chemical functionalities into a three-gene construct that confers improved tolerance for these inhibitor compounds. The information gleaned from this study will be useful for scientists who wish to develop strains of bacteria that can generate new biofuels more efficiently and for longer periods of time.

Other Epicentre products cited include the End-It™ DNA End-Repair Kit for end-polishing of size-selected, gel-purified DNA; and the FosmidMAX™ DNA Purification Kit to purify DNA from the metagenomic fosmid clones.

ResearchBlogging.orgSommer, M. et al. (2010). A functional metagenomic approach for expanding the synthetic biology toolbox for biomass conversion Molecular Systems Biology, 6 DOI: 10.1038/msb.2010.16

Thursday, July 29, 2010

Solutions for problematic plant templates

Recently, one of our customers (Millie Burrell of Texas A&M University) thanked us for helping her resurrect some DNA templates that she was on the “verge of abandoning due to lack of quality DNA and subsequent PCR amplification for downstream sequencing.” Through the use of the QuickExtract™ Seed Solution and PlantAmp™ PCR System, she was able to obtain PCR results for DNA extracted from Caulanthus amplexicaulus, a little-known wild relative of Arabidopsis. According to Millie, this plant is a “rare and endangered species that grows on serpentine (ultramafic) soils known for low macronutrient levels and conventionally toxic levels of heavy metals.” The QuickExtract Seed protocol uses up to 10 mg of ground/fragmented seed in an 8-minute protocol that is suitable for high-throughput workflows. The PlantAmp PCR System is formulated to optimize PCR amplification from samples containing polyphenols and other PCR inhibitors, in a convenient premix format.

Photo credit: joedecruyenaere

Wednesday, July 14, 2010

Fosmid cloning: Alive and kicking

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 isolated from E. coli, while protecting its own DNA by site-specific phosphorothioation. They located the gene in S. enterica by screening a genomic library created using the CopyControl™ Fosmid Library Production kit. The authors screened the fosmid libraries using degenerate gene cluster–specific primers to locate the genes responsible for phosphorothioation and also to perform mutational analysis. Their results establish a biological role for phosphorothioation activity, and suggest that DNA S-modification may act not only as protective system against bacteriophage infection, but also as an epigenetic signal for new biological functions.

In another study by Lucker et al.2, the CopyControl Kit was used to reconstruct the complete genome of Candidatus Nitrospira defluvii (Ca. N. defluvii) from a metagenomic fosmid library prepared from an activated sludge enrichment culture. The immense ecological and technical significance of Nitrospira contrasts with the scarce knowledge about these bacteria. Except for one 137-kb contig, genomic sequences from Nitrospira have not been obtained yet. This situation has been highly unsatisfactory because deeper insight into the biology of these elusive nitrate oxidizing bacteria is crucial for a better understanding of nitrogen cycling in natural and engineered systems. On the basis of this first-deciphered Nitrospira genome and experimental data, the authors show that Ca. N. defluvii differs fundamentally in its enzymatic repertoire and metabolic pathways from all other known nitrifying bacteria. The current study provides valuable insights into the evolution of nitrite oxidation.

ResearchBlogging.org1. 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
2. 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

Friday, July 9, 2010

When is “direct PCR” not direct?

We’ve seen a few companies promoting “direct PCR” products, where a sample can be added directly to a PCR mixture without first extracting DNA. This seems like a good idea in theory, but the reality is not as simple--especially for samples that contain PCR inhibitors.

Therefore, companies that offer kits for direct PCR have had to lengthen these procedures to contend with inhibitors. For example, one kit requires NaOH treatment for 10 minutes, followed by neutralization with Tris-HCl. A large PCR volume is needed and an additive must be included in the gel loading buffer prior to electrophoresis. A second direct PCR kit also requires a large PCR volume and an additive in the loading buffer. In addition, since the polymerase is not Taq-based, annealing temperature must be carefully optimized. For this kit, alternate protocols are offered for challenging templates or multiplex PCR consisting of adding Dilution Buffer and Additive, followed by vortexing, centrifugation, two incubations, and a second centrifugation.

Both kit protocols described above are based on processing animal tissue samples. For plant tissue, even more steps are needed to remove inhibitors.

In contrast, Epicentre’s QuickExtract™ products offer a rapid, efficient method to extract nucleic acids from virtually any sample for PCR-based assays, with most samples being processed in only 8 minutes. Our guaranteed FailSafe™ PCR System ensures reliable PCR results, the first time and every time. Together, these products provide a simpler and more effective method than direct PCR.

Special offer: Order the FailSafe PCR PreMix Selection Kit and get 5 ml of any QuickExtract product free.

Tuesday, July 6, 2010

Gene expression analysis from Ribo-Zero™-treated RNA

The Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) was compared to a competitive kit's performance in removing rRNA from total RNA samples that were used for RNA-Seq. Both kits performed equivalently for intact RNA samples, while the Ribo-Zero Kit performed better than the competitive kit for partially degraded RNA samples.

A

B

C

Consistent gene expression data from Ribo-Zero™-treated RNA. Intact and partially degraded Universal Human Reference RNA (UHRR) was treated with either the Ribo-Zero Kit or a competitive kit. The rRNA-depleted RNAs were then used to prepare cDNA libraries that were sequenced on an Illumina® GAII sequencer.  A) Number of genes detected in libraries prepared from intact or partially degraded, rRNA-depleted UHRR. B) Correlation of expression levels for intact RNA. C) Correlation of expression levels for partially fragmented RNA. RPKM, reads per kilobase of exon model per million mapped reads.

(Click images to enlarge them.)

Friday, June 25, 2010

Functional metagenomics reveals mechanisms of antibiotic resistance

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 resistance based on the ability to grow these microbes can fall short in locating these resistance genes, primarily due to the inability to cultivate some of these bacteria. Functional metagenomics, which involves inserting large fragments of foreign DNA into E. coli and assaying the resulting clones for expressed functions, allows the study and isolation of various activities encoded by genes from microbes that are otherwise uncultivatable.

Among 13 antibiotic-resistant clones, the authors isolated two genes that encode novel bifunctional proteins. One putative bifunctional protein confers resistance to ceftazidime and contains a natural fusion between a predicted transcriptional regulator and beta-lactamase. The ability to archive these activities in a genomic library enhances their future study under many different conditions.

ResearchBlogging.orgDonato, 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

Tuesday, June 22, 2010

Superior ribosomal RNA (rRNA) removal for RNA-Seq

Epicentre recently launched the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat). The kit removes >99% of the 28S, 18S, and 5.8S and >95% of the 5S rRNA from both intact and partially degraded human, mouse, and rat total RNA preparations. The core procedure takes less than an hour; the rRNA-depleted samples can be purified either by ethanol precipitation or a column-based method. The kit provides an ideal solution for users who are preparing RNA-Seq libraries from human, mouse, or rat total RNA, especially from compromised samples such as formalin-fixed, paraffin-embedded (FFPE) tissue.

The Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) significantly improves RNA-Seq results. Intact and partially degraded Human Reference RNA was treated with either the Ribo-Zero Kit or a competitive rRNA removal kit. The rRNA-depleted RNA was then used to prepare RNA-Seq libraries that were sequenced on an Illumina® GAII sequencer.
Total RNA Sample rRNA Removal Kit % Ribosomal RNA Sequenced
Intact Human Reference RNA Ribo-Zero™ Kit 1.4%
Intact Human Reference RNA Competitive Kit 18.4%
Partially Degraded Human Reference RNA Ribo-Zero™ Kit 2.1%
Partially Degraded Human Reference RNA Competitive Kit 63.3%

Friday, June 18, 2010

Get rid of the small stuff

We have received quite a few enquiries from our customers on how to remove low molecular-weight (MW) fragments from the Nextera™ library following tagmentation and limited-cycle PCR. Based on extensive R&D testing, we’ve found that AMPure® XP beads from Beckman Coulter work very well in removing fragments below 350 bp. The 0.7X bead concentration is effective at removing lower MW fragments (<350 bp) from Nextera libraries.

Note: The 350-bp cut-off point refers to fragments below 350 bp; the actual insert size (of the genomic DNA) is approximately 250 bp (Nextera Roche-Compatible) or 215 bp (Nextera Illumina-Compatible). The reason for the difference is due to the adaptor sequences on the genomic DNA fragments. The Roche library fragments contain 98 bp of adaptor/transposon-end sequences, and the Illumina library fragments contain 135 bp of adaptor/transposon-end sequences. These additional adaptor/transposon-end sequences make the genomic DNA fragments appear bigger than their actual size.

Below we show two Bioanalyzer traces of Nextera libraries, one purified using Zymo DNA Clean and Concentrator™, the other using AMPure XP beads from Beckman Coulter.

Note: Zymo or AMPure clean-up was performed after limited-cycle PCR, just prior to loading the libraries on emulsion PCR or bridge PCR.

(click to enlarge)
Red: Illumina-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 9 cycles PCR, Zymo DNA Clean & Concentrator-5.
Blue: Illumina-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 9 cycles PCR, Agencourt AMPure XP beads (0.7X).


(click to enlarge)
Red: Roche-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 15 cycles PCR, Zymo DNA Clean & Concentrator-5.
Blue: Roche-compatible Nextera library, 50 ng Lambda DNA, HMW Buffer, 15 cycles PCR, Agencourt AMPure XP beads (0.7X).

Tuesday, June 15, 2010

Visit our poster at the CHI Beyond Sequencing meeting

Epicentre will be presenting a poster titled “Advances in Next-Generation Sequencing Library Preparation” at the CHI Beyond Sequencing meeting, to be held June 22-23 in San Francisco, CA. We will describe our new technology for preparing Roche and Illumina-compatible libraries for next-generation sequencing (NGS) from only 50 ng of DNA, in less than 2 hours. We will also discuss new applications of our Nextera™ technology, including methyl-Seq, and also products for rRNA reduction for NGS, and mRNA/small-RNA NGS library prep kits. Please stop by our poster for additional information, or contact us by e-mail if you're not able to attend the meeting.

Tuesday, June 8, 2010

Library preparation for ChIP-Seq

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 End Repair Kit, Exo-Minus Klenow, and the Fast-Link™ DNA Ligation Kit, to end-repair, A-tail, and ligate Illumina Genomic Adaptors to sheared DNA. The end-tagged DNA was PCR-amplified, and sequenced using an Illumina Genome Analyzer II. The researchers report a solid smear of DNA at the 160-230 bp size range, and a secondary smear of less intensity (around 400 bp), which they attributed to dinucleosomal DNA. The sequencing data were used to analyze the number and frequency of epigenomic changes in the prefrontal cortex neurons, with important implications for a variety of neurodevelopmental disorders.

ResearchBlogging.orgCheung, 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

Friday, June 4, 2010

From cows to chickadees: BuccalAmp™ Kits in animal genomics

Animal subjects have benefited from Epicentre's BuccalAmp™ DNA Extraction Kit, a single-tube system for rapid extraction of PCR-ready DNA. Buccal cell sampling offers a quick, easy, economical, and less invasive alternative to other methods of acquiring DNA from animals. Previous reports have described the use of BuccalAmp Kits to examine quantitative trait loci affecting milk production and reproduction in commercial dairy cattle at the USDA, and to study multiple intestinal neoplasia in mice (Epicentre Forum 9-2, 251K PDF).

Researchers at the US Geological Survey [Handel, CM et al., (2006) Wildlife Soc Bull 34:1094; abstract] used the BuccalAmp Kit to isolate DNA from buccal cells of adult and nestling chickadees, and compared the results to those obtained with blood draws.  They highly recommended the use of buccal swabs as a rapid, noninvasive technique for sampling avian genomic DNA in small birds or any birds in which blood testing may be difficult or stressful. http://www.jstor.org/pss/4134320

Recently, the BuccalAmp Kit was used in a study of GM1 gangliosidosis in Japanese Shiba dogs (Chang, H-S et al., (2010) J Vet Diagn Invest 22:234; abstract]. DNA was isolated from buccal cells or blood/tissue samples, as well as from FTA cards. The target sequences were amplified and detected by real-time PCR using Taqman® probes. The BuccalAmp kit was quicker, simpler to use, and provided more reliable results than blood or tissue sampling.

Epicentre provides several options for collecting buccal samples; visit our buccal swabs and brushes product page for more information.

Tuesday, June 1, 2010

ASM 2010: A good week for Epicentre

Epicentre employees were busy last week at the ASM 2010 General Meeting, with a lot of interest in products for DNA purification and next-generation sequencing. A significant amount of the posters presented at the meeting, as well as the conference sessions, were devoted to metagenomics. The growing popularity and declining costs for next-generation sequencing services have facilitated metagenomic analysis of microbial populations from a diverse set of environments--from hot springs to human armpits.

We recently introduced the Meta-G-Nome™ DNA Isolation Kit, designed for environmental samples. However, we are always interested in other applications for the kit, and we encourage any of our readers who are interested in other applications for the kit to take our online survey. By doing so, you'll receive a special 50% evaluation discount on the kit (offer currently limited to U.S. customers only).

Monday, May 24, 2010

Purifying bacterial DNA from contaminated food samples

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 researchers isolated DNA from 25 food samples including mixed salad, minced meat, nonpasteurized milk, carrots, turnips, cabbage, lettuce, onion, pumpkin, and tomatoes. They developed TaqMan® qPCR assays using gene-specific probes to detect the Y. pseudotuberculosis ail gene, and distinguish it from related Y. enterocolitica serotypes. Whereas 6 of the analyzed food samples were positive for Y. pseudotuberculosis as indicated by PCR, all 25 were negative when analyzed by the traditional culture method.

ResearchBlogging.orgLambertz, 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

Tuesday, May 18, 2010

Structural differences between Marburg and Ebola viruses

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 to investigate differences in RNA secondary structures between MARV and EBOV. They analyzed the structure of the MARV 3’-noncoding region and its influence on VP30. VP30 is an RNA binding protein which acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription.

The researchers choose the AmpliScribe system because it can efficiently transcribe RNA from limited amounts of DNA (as low as 1 ng). The AmpliScribe High Yield Transcription Kits can produce >20-fold more full-length RNA (both short and long transcripts) than conventional in vitro transcription reactions.

ResearchBlogging.orgEnterlein, 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

Friday, May 14, 2010

Visit Epicentre at ASM 2010 in San Diego, CA

We’re gearing up for our annual pilgrimage to the American Society for Microbiology (ASM) General Meeting, May 23-27. Searching the abstracts, we noted many posters and presentations citing Epicentre’s unique products for molecular biology research. Posters and student seminars have been presented at previous ASM meetings featuring EZ-Tn5™ transposon mutagenesis, large-insert cloning with CopyControl™ products, PCR using the renowned FailSafe™ PCR System, DNA purification from a variety of sources using the MasterPure™ kits, and many other Epicentre products.

For those of you attending ASM 2010, you can search the abstracts for topics of interest and citations of Epicentre products.  Also, visit our exhibit (#122) to learn more about:
  • Creating metagenomic libraries from water, soil, compost, and air samples;
  • Nextera™ Sample Prep Technology for next-generation sequencing of microbial and metagenomic DNA libraries;
  • In vivo and in vitro transposition systems for DNA recombination and engineering.
Technical support and marketing staff will be present to answer your questions and discuss our new products. And, of course, we’ll have our booth stocked with ever-popular giveaway items!

We hope to see you at ASM 2010! Feel free to leave a comment and let us know if you’re attending.

Tuesday, May 11, 2010

Isolation of RNA from breast cancer FFPE tissues

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 shift in raw CT values compared to FF tissues. However, the authors demonstrate that proper normalization of the expression data can compensate of the effects of RNA degradation. The authors used the MasterPure™ RNA Purification Kit to isolate RNA from FFPE tissue slices and examined expression levels of reference and breast cancer prognosis–related genes using TaqMan® low-density arrays. Based on their analysis, they make recommendations for proper normalization strategies when using FFPE samples in qRT-PCR analysis. The authors conclude:

Nevertheless, careful selection of candidate biomarkers should be made: those genes that show no correlation between FF and FFPE should not be included in molecular tests for clinical use based in FFPE samples. Moreover, in order to guarantee reliable results in gene expression measurements, we strongly encourage performing preliminary studies, with the aim of discarding noncorrelated genes.

ResearchBlogging.orgSánchez-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 : 10.2144/000113388

Thursday, May 6, 2010

Nextera™ library fragment size range

Another common question regarding our Nextera™DNA Sample Prep Kits for next-generation sequencing:

The Nextera library fragments are too small. Is there a better way to control the DNA fragment size?

Nextera DNA Sample Prep kits include two buffers, Low Molecular Weight (LMW) and High Molecular Weight (HMW), for generating two size classes. In general, with the Nextera Illumina-Compatible Kit, the LMW buffer will produce fragments from 150 to 600 bp (peak around 200 bp). The HMW buffer will produce fragments from 175 to 700 bp (peak around 250 bp). The final fragment size includes approximately 100 bp of adaptor/transposon end sequence, so the actual genomic fragment is smaller than the apparent MW of the library. It is normal for the fragment MW to vary slightly. This is commonly a result of DNA type, quality, and purification and quantitation methods used.

The quality of the starting DNA is critical. Contaminants such as protein and RNA may inhibit the Nextera tagmentation reaction if present in the DNA preparation. Therefore, it is important to start with highly pure  DNA. We also recommend using HMW buffer if small fragment size is an issue. The current protocol has been developed with 50 ng of DNA. If the fragments are too small, we recommend adding slightly more DNA. Changing the temperature or reaction time is not a robust or reproducible way to control fragment size. Finally, if a narrow MW distribution is required, it may be necessary to perform a size-selection step (gel, AMPure® beads, SPRIWorks®, etc.) after the limited-cycle PCR.

Monday, May 3, 2010

Transposon mutagenesis identifies a novel toxin regulatory locus in Clostridium perfringens

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 resistance to its targeted host cell. Clostridium perfringens is a Gram-positive, obligate spore-forming anaerobe. Its growth characteristics pose considerable obstacles to the use of transposon mutagenesis in the search for genes associated with virulence factors.

The authors used the pMOD-2 Transposon Construction Vector to build an erythromycin-resistance transposon using the erm gene from the E. coli-C. perfringens shuttle vector pJIR751. The Transposome complex was built using standard procedures and transformed using a Biorad Gene Pulser™ electroporator. Using the EZ-Tn5 system, the researchers were able to locate a mutant that disabled the toxin-13 regulatory locus (determined to be similar to the Agr locus of Staphylococcus aureus). They also reported that the generation of mutant Clostridia using the EZ-Tn5 Transposomes was much more efficient than other random mutagenesis methods.

If you're considering the EZ-Tn5 system for bacterial mutagenesis, visit the EZ-Tn5 Transposomes citation page for information on your species of interest.

ResearchBlogging.org*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

Thursday, April 29, 2010

Identification of suitable hosts for small-molecule functional metagenomics

The majority of soil-dwelling bacteria cannot be cultured with standard microbial culture methods, and represent an untapped reservoir of novel small molecules that are key components of biosynthetic pathways. Functional metagenomics is one approach to solving this problem; however, screening methods are limited by their dependence on a host organism to facilitate the expression of genes from environmental DNA (eDNA).  To overcome the limitations of relying on a single host organism, Craig et al.* examined six unique Proteobacteria hosts for functional metagenomic screening. To construct  soil eDNA libraries, they blunt-ended the isolated DNA with the End-It™ DNA End-Repair Kit, ligated it into a cosmid vector, packaged the DNA using MaxPlax™ Lambda Packaging Extracts, and transformed TransforMax™ EC100 Electrocompetent E. coli. The eDNA libraries were then used to transform each of the six hosts being studied, and the resulting libraries were screened for antibacterial activity, or altered pigment or morphology. Cosmid DNA from selected colonies was electroporated into TransforMax™ EPI300 Electrocompetent E. coli, and each cosmid clone was analyzed by 454 sequencing.

From this initial broad-host-range functional metagenomic study, Craig et al. demonstrate that some surrogate expression hosts will be better suited to using foreign genetic material than others. They conclude that continued exploration of phylogenetically diverse bacteria as hosts for functional metagenomic screening is likely to identify additional strains that will be suitable for eDNA functional metagenomics.


ResearchBlogging.org*Craig, J. et al. (2010). Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria Applied and Environmental Microbiology, 76 (5), 1633-1641 DOI: 10.1128/AEM.02169-09

Tuesday, April 27, 2010

Labeling RNA for gene expression analysis

Here are answers to questions that we commonly receive regarding the TargetAmp™ Antisense RNA (aRNA) Amplification Kits.

Can I use the TargetAmp Kits for producing fluorescent-labeled aRNA?
Yes. The TargetAmp 1-Round and the TargetAmp 2-Round Aminoallyl-aRNA Amplification Kits produce aminoallyl-aRNA (AA-aRNA), which can be readily labeled with a fluorescent dye conjugated to N-hydroxysuccinimide (NHS). 

What are the advantages of aminoallyl-based (indirect) labeling over direct incorporation of a labeled NTP?
Aminoallyl-UTP is more efficiently incorporated into the aRNA during the in vitro transcription reaction, compared to labeled nucleotides. Additionally, the conjugation of an amine-reactive NHS ester of biotin or a fluorescent dye to AA-aRNA can be a less expensive method to label the target compared to direct incorporation of labeled nucleotides. 

What are the advantages of direct labeling of aRNA?
Labeling by direct incorporation is faster compared to the aminoallyl-based procedure. Conjugation of AA-aRNA to a dye- or biotin-NHS ester after in vitro transcription requires a 1-hour incubation, and an additional clean-up step, compared to direct incorporation. Further, labeling aRNA by direct incorporation does not require the use of toxic reagents (such as DMSO), which are used in the indirect labeling protocol.

To find the TargetAmp Kit that’s right for your application, please see our selection guide.

Friday, April 23, 2010

EZ-Tn5™ Transposomes help reveal virulence factors in Acinetobacter baumanii

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. Unfortunately, very little is known about the genetics of this organism, which hinders the study of pathogenic factors in A. baumanii infection. In a recent study, Jacobs et al.* describe the use of the EZ-Tn5™ Transposome in inactivation of the Phospholipase D gene in various strains of A. baumanii, and report that the creation of a polar mutation in this locus reduces the pathogenicity of the bacterium. Inactivation of Phospholipase D showed reduction of overall A. baumanii bioburden in the blood, heart, and liver in a murine model, but did not reduce the bioburden in the lungs. The utility of the EZ-Tn5 Transposome system to develop genetic systems for novel or poorly studied microbes is once again demonstrated.

ResearchBlogging.org*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

Tuesday, April 20, 2010

Complete Genomics sequencing platform built on Epicentre’s enzymes

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.* The researchers sequenced three human genomes, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome.

The combinatorial probe-anchor ligation (cPAL) chemistry relies on the formation of DNA concatamers (termed “nanoballs”) that are clonally amplified from circular templates (see Complete Genomics workflow summary). The templates are prepared using Epicentre’s CircLigase ssDNA Ligase. In addition, the library preparation procedure used:
ResearchBlogging.org
*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

Thursday, April 15, 2010

FailSafe™ PCR System earns praise

It's always nice when customers appreciate our products. It's even better when they make a video telling the world about why the product performed so well.

Alan M at Benchfly describes his experience with FailSafe™ Buffer D in the clip below. No, we didn't pay him a dime :) We appreciate his comments and, of course, we agree with his assessment of why the FailSafe System is the best product for PCR of challenging and routine templates. Don't just take our word for it, though--the FailSafe PCR System is supported by over 100 publications.

Note: We recommend that customers always optimize their PCR conditions for each new template, using the FailSafe PCR PreMix Selection Kit.

Tuesday, April 13, 2010

CHI XGen Congress session on Nextera™ technology

Epicentre presented a breakfast session on Nextera™ technology and its applications at the CHI XGen Congress (March 15-19, 2010). We are pleased to make a video recording of the session available through SciVee. The presentation is approximately 27 minutes long.

Friday, April 9, 2010

Save big on library prep for Roche GS FLX

As many users of Roche 454 sequencers have moved to the newer GS FLX Titanium chemistry, we are now offering big discounts on Nextera™ DNA Sample Prep Kits for the original GS FLX chemistry (Cat. # FL09115 and FL091120).

Let us know if you’re using the original GS FLX reagents by leaving a comment here. If you’d like to try the Nextera library prep method—which offers a streamlined, rapid workflow and low input DNA requirements—this is your chance to save money as well as time. Please contact us by e-mail to obtain a discounted price: Nextera {at} epibio {dot} com

Tuesday, April 6, 2010

Transposomics made EZ

EPICENTRE’s EZ-Tn5™ Transposomes (the synaptic complex between  transposon DNA and the hyperactive EZ-Tn5 Transposase) have been used for generating bacterial mutation libraries, strain development, and creating knockout/knock-ins since 1999. The easy-to use Transposomes have been used in the development of transposition libraries in over 65 microbial species (both Gram-positive and Gram-negative), and even in yeast and trypanosomes. Our citations page lists publications grouped by the organism studied.

The ready-to-use Transposomes contain kanamycin- or trimethoprim-resistance cassettes. Additional Transposomes can be created easily, using simple molecular techniques  with the EZ-Tn5 pMOD series of transposon construction vectors and hyperactive EZ-Tn5 Transposase. We also provide a comprehensive introduction to transposon methods and product selection guide, and additional documentation (114K PDF) for the preparation and use of custom Transposomes in many bacteria.

Tuesday, March 30, 2010

Nextera™ tagmentation of soil metagenomic DNA

High-throughput sequencing has greatly facilitated metagenomic analysis of environmental samples. Epicentre recently introduced the Meta-G-Nome™ DNA Isolation Kit for use with water, soil, and compost samples. In this analysis, we isolated metagenomic  DNA from garden soil and performed the tagmentation reaction according to the Nextera™ protocol. The isolated DNA yielded tagmentation products in the expected size range, compared to a control that was also tagmented according to the Nextera protocol (Fig. 1).

Figure 1. Nextera tagmentation of soil metagenomic DNA. Garden soil that had been stored for 6 months at 4°C was used as starting material. DNA was isolated according to the protocol for the Meta-G-Nome DNA Isolation Kit from 1 g of soil, using a final elution volume of 20 µl. The metagenomic DNA and a control (lambda DNA) were tagmented according to the Nextera protocol, and the tagmentation products were analyzed on a 1% agarose gel. Lane 1, 5 µl of metagenomic DNA before tagmentation; lane 2, tagmented lambda DNA (50 ng); lane 3, tagmented metagenomic DNA (10 µl used for tagmentation); lane 4, tagmented metagenomic DNA (17 µl used for tagmentation); lanes M, 1-kb DNA ladder.


The tagmented metagenomic DNA and lambda DNA were then used as templates in PCR with either Nextera primers (Roche FLX-compatible), or primers for the 16S rRNA gene (Fig. 2). PCR with Nextera primers yielded amplification products in the expected size range (Fig. 2, lanes 2 and 3), and metagenomic DNA yielded the expected 16S rRNA amplification product (Fig. 2, lanes 5 and 6).

Figure 2. PCR of tagmented metagenomic DNA. PCR was performed using tagmented metagenomic DNA and lambda DNA as templates, with Nextera FLX-compatible primers (lanes 1-3). Metagenomic DNA was also amplified using 16S rRNA gene primers as a control (lanes 4-6). PCR products were cleaned up using Zymo columns prior to electrophoresis in a 1% agarose gel. Lanes 1 and 4, no-DNA control; lane 2, PCR of tagmented lambda DNA; lane 3, PCR of tagmented metagenomic DNA; lanes 5 and 6, PCR of metagenomic DNA (undiluted and 1:10 diluted template, respectively).


If you’re interested in purifying metagenomic DNA from soil, compost, or water samples, and would like to evaluate the Nextera DNA Sample Prep Kits for next-generation sequencing, please fill out a short survey.