EpiCentral

Transcriptome-wide discovery of circular RNAs in Archaea

2012-01-30T08:35:00.000-08:00

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

Transcriptional profiling of an enterotoxigenic E. coli isolate

2012-01-25T08:08:00.000-08:00

Ribo-Zero Kits have rapidly established themselves as the method of choice for depleting ribosomal RNA (rRNA) for RNA-Seq studies. Their utility extends to microarray-based analysis of gene expression. Sahl et al. describe the use of RNA-Seq and microarray analysis to study the effects of chemical signaling factors in the pathogenesis of enterotoxigenic Escherichia coli (ETEC). This organism is an important pathogenic variant (pathovar) of E. coli in developing countries and is associated with significant morbidity and mortality rates.

The research focused on providing various stimulants to the cells in culture followed by RNA extraction to measure the up- or down-regulation of the enterotoxins being produced by the cells. The Ribo-Zero Gram-Negative Kit was used to remove rRNA from the total RNA preparations, simplifying data analysis from RNA-Seq libraries and microarray expression analysis. RNA-Seq results demonstrated bile salts regulate many virulence factors; one of the most differentially expressed genes in the presence of bile is a unique plasmid-encoded AraC-like transcriptional regulator (peaR).

The authors conclude: "These results provide transcriptional targets and putative mechanisms to better understand the global regulatory networks and virulence expression in this important human pathogen."

Sahl, J. and Rasko, D. (2012). Analysis of the global transcriptional profiles of enterotoxigenic Escherichia coli (ETEC) isolate E24377A Infection and Immunity DOI: 10.1128/IAI.06138-11

Genomic study of deadly bovine pathogen faciliated by QuickExtract Bacterial Kit

2012-01-12T14:16:00.000-08:00

Mannheimia haemolytica is a Gram-negative bacterium associated with bovine respiratory disease complex. During stress, such as a viral infection and/or transportation to the feedlot, the bacterium transforms from benign to deadly, and causes a disease called shipping fever, resulting in losses of more than $1 billion annually in the U.S. Despite its economic importance, there are no specific and accurate genetic markers for this disease.

Researchers at Washington State University (Lawrence PK et al., BMC Genomics 2010, 11:535) performed a three-way comparison between the genomic sequences of three strains of M. haemolytica from cattle and domestic sheep. They extracted total genomic DNA using the QuickExtract Bacterial DNA Extraction Kit, and prepared genomic libraries for sequencing on a Genome Sequencer FLX (Roche). At 20X sequence coverage, the authors identified a number of genes that are unique to each strain. In addition, many high-confidence single nucleotide polymorphisms (hcSNPs) were identified, which will be used to design new arrays to study variation across strains and potentially aid in understanding gene regulation and mode of action. Additional virulence factors included a previously unknown type III secretion system, and CRISPR loci that indicates the potential resistance of M. haemolytica to superinfection by phages. The study also identified various adhesins, containing protein cleavage domains, that could potentially serve as effective vaccine targets.

Visit Epicentre at the International PAG XX Conference

2012-01-06T11:01:00.000-08:00

Epicentre will be attending the International Plant and Animal Genome (PAG) XX Conference, to be held from January 14-18 in San Diego, CA. Stop by Booth #209 to learn more about our products for RNA-Seq library preparation, plant and animal genomics, and to receive special discounts on a variety of Epicentre products.

In addition, we will be presenting the following posters:

We hope to see you at the conference! If you're not attending, and would like more information about the products highlighted at the conference, please contact us by e-mail or call 1 (800) 284-8474 within the US.

Large-insert cloning aids study of dinoflagellate species

2011-12-22T07:51:00.000-08:00

Large-insert cloning products have long been a mainstay of Epicentre's product line and are critical for the study of gene expression and interactions. Recently, Jaeckisch et al. described making three kinds of libraries (cDNA, fosmid, and BAC) for characterizing the marine dinoflagellate Alexandrium ostenfeldii.

Many dinoflagellate species are notorious for the toxins they produce, as well as ecological and human health consequences associated with harmful algal blooms (HABs). One way to study these otherwise toxic compounds is to build their synthesis genes into a large-insert-capable, low-copy cloning vector, transform into a suitable host cell, and then perform the desired studies in such a way that the genes will not have a negative affect on the host. The genes for the toxins cited in the publication (macrocyclic imine toxins, described as spirolides), were inserted into the pCC1FOS vector and transfected into the TransforMAX™ EPI300 host strain for further study. Further, genomic DNA from A. ostefendii was prepared for cloning into the pIndigoBAC-5 HindIII Cloning-Ready vector and transformation into the TransforMAX EC100 host.

A total of 384 BAC clones were obtained with insert sizes ranging from 50 to 150 kb, which provided sufficient coverage to allow elucidation of the whole genome sequence and some comparative sequence data with the marine dinoflagellate H. triquetra. The authors used the sequence information obtained from the BAC and CopyControl fosmid libraries to investigate spliced leader (SL) trans-splicing and mRNA transposition mechanisms. They characterized the genome using selected clones, using a combination of transcriptomic data and random genomic clones. Examination of SL sequences revealed similar features as in other dinoflagellates, including other Alexandrium species. SL sequences in decay indicate frequent retro-transposition of mRNA species. The transposition observed in these genes probably contributes to overall genome complexity by generating additional gene copies.
The authors conclude:

The genomic sequence survey (GSS) represents the largest genomic data set of a dinoflagellate to date...The large portion of repetitive sequences and the organization within the genome is in agreement with several other studies on dinoflagellates using different approaches. It remains to be determined whether this unusual composition is directly correlated to the exceptionally [sic] genome organization of dinoflagellates with a low amount of histones and histone-like proteins.

Jaeckisch, N. et al. (2011). Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii PLoS ONE, 6 (12) DOI: 10.1371/journal.pone.0028012

What's new in the ScriptSeq v2 kit?

2011-12-12T15:11:00.000-08:00

Recently, Epicentre launched the ScriptSeq™ v2 RNA-Seq Kit for preparing directional, ligation-free RNA-Seq libraries in 4 hours. This kit offers several advantages over the previous version:

Transcript coverage is improved and GC bias is reduced by an improved terminal-tagging oligo (TTO).
Less input RNA required: Libaries can be prepared from as little as 500 pg of rRNA-depleted or poly(A)-enriched RNA.
A streamlined protocol and premixed reagents make the kit easier to use and require fewer pipetting steps.
A signifcantly lower price compared to the original kit.

The original ScriptSeq Kit will be available for a transition period, to allow existing customers to finish important projects. We expect customers will find that the new ScriptSeq v2 Kit provides better performance at a lower price.

Role of Piwi proteins in mammalian transposon silencing

2011-12-09T11:40:00.001-08:00

In a recent Nature publication, Reuter et al. report on the study of certain small RNAs that affect the fertility of male mice. Piwi-interacting RNAs (piRNAs) act together with Piwi proteins Mili (also known as Piwil2) and Miwi (also known as Piwil4) in a genome defense mechanism that initiates transposon silencing via DNA methylation in the mouse male embryonic germ line. This silencing depends on the participation of the Piwi proteins in a slicer-dependent piRNA amplification pathway and is essential for male fertility.

The researchers cite the use of Epicentre’s Ribo-Zero™ Kit (Human/Mouse/Rat) to remove interfering ribosomal RNA from the experimental matrix, allowing closer study of the interactions between piRNAs. They also used the ScriptSeq™ mRNA-Seq Kit for an unusual application: sequencing small RNAs. Under normal circumstances, small RNAs <50-60 nucleotides are better suited for Epicentre's ScriptMiner™ Small RNA-Seq Kit, due to potential issues with the ScriptSeq method at the 5’ end of small RNAs. These results demonstrate that the ScriptSeq Kit has the ability to prepare RNA-Seq libraries from RNA types other than mRNA, including small RNAs.

Reuter, M. et al. (2011). Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing Nature, 480 (7376), 264-267 DOI: 10.1038/nature10672

Transposome-based RNA-Seq library construction from low input amounts of RNA

2011-12-02T07:55:00.001-08:00

A collaborative effort between Epicentre and the HudsonAlpha Institute for Biotechnology resulted in the development of two novel transposon-based methods for RNA-Seq library preparation. The technique, called Tn-RNA-Seq, can use double-stranded cDNA created from rRNA-depleted RNA to prepare an Illumina^® sequencing library using only two enzymatic reactions. The researchers generated high-quality RNA-Seq libraries from as little as 10 pg of mRNA (~1 ng of total RNA) with this approach.

They also present a strand-specific RNA-Seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and Endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. These directional RNA-Seq libraries maintained the same quality as the nondirectional libraries, while showing a high degree of strand specificity (99.5% of reads mapped to the expected genomic strand).

A key benefit of the Tn-RNA-Seq technique is the ability to use extremely low amounts of RNA to prepare high-quality libraries. All six libraries generated using 10 ng to 10 pg of mRNA had at least 72% of aligned reads map to known transcripts, while the library made from 1 pg of mRNA had 62% of aligned reads map to known transcripts. Library complexity was found to be high for all libraries except for the library constructed with 1 pg of mRNA. In general, Tn-RNASeq libraries made with 10 pg or more of mRNA (about 50 cell equivalents) exhibited consistent quality measures. For all libraries except for the library made with 1 pg of mRNA, the rank correlations remained very high (>0.96) indicating highly consistent and reproducible library formation. The directional Tn-RNA-Seq libraries retained the same level of “strandedness” during sequencing compared to libraries made using standard adaptor-ligation methods.

The authors concluded that high-quality RNA-Seq libraries can be constructed efficiently from low input amounts of RNA using the Tn-RNA-Seq methods, and that the procedure is suitable for high-throughput or automated workflows.

Gertz, J. et al. (2011). Transposase mediated construction of RNA-seq libraries Genome Research DOI: 10.1101/gr.127373.111

Ribosomal RNA depletion produces improved whole-transcriptome RNA-Seq results

2011-11-18T14:40:00.001-08:00

In a recent PLoSOne publication, Huang et al. compared the use of the Ribo-Zero™ rRNA Removal Kit (Human/Mouse/Rat) to the RiboMinus™ rRNA Removal Kit (Life Technologies) and poly(A) enrichment in preparation of RNA-Seq libraries. They studied two types of mouse tissue samples: differentiated embryonic stem cells (CCE) and fetal head (FH).
The researchers compared samples that were treated by rRNA removal and RNA chemical fragmentation procedures on the basis of gene expression analysis, quality of sequence data, number of reads, retention of sequence tags, and any biases noted in the sequencing. Some notable conclusions from this study:

RiboMinus treatment may require two rounds of rRNA depletion whereas Ribo-Zero treatment only requires round of depletion.
The use of RNA hydrolysis and Ribo-Zero treatment result in more efficient removal of rRNA, as compared to RNA hydrolysis and RiboMinus treatment. The researchers also observed an increase in uniquely matching (36%-54%) and multiple matching tags (28%-30%). Interestingly, tags with no match in the genome did not change (14%-21%.)
In comparison to the RiboMinus preparation, Ribo-Zero treatment results in RNA-Seq libraries with more tags in the 5′ and 3′ ends of small genes compared to the body of the genes. The coverage of other gene sizes was largely unchanged between the two rRNA depletion methods.
RNA-Seq libraries prepared from ribo-depleted (e.g., Ribo-Zero-treated) RNA have significant advantages over poly(A)-enriched RNA for detecting macro ncRNAs.

Huang, R. et al. (2011). An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs PLoS ONE, 6 (11) DOI: 10.1371/journal.pone.0027288

Medicinal leeches, MasterPure RNA Kits, and metatranscriptomics

2011-11-09T13:26:00.000-08:00

The overwhelming majority of microorganisms in existence have not been cultured in the laboratory. Even the most abundant and metabolically active members of the microbial community can be challenging to cultivate. Next-generation sequencing is providing insight into the metabolic relationships within microbial communities by sequencing the metatranscriptome.

Bomar et al. report on the gut microbiome of the medicinal leech Hirudo verbena. They found two symbionts in the digestive tract: i) a Rikenella-like bacterium; and ii) Aeromonas veronii. After a blood meal, the largest compartment of the digestive tract (the crop), stores ingested blood for months between feedings. Water and salts are removed from the ingested blood meal, creating a viscous intraluminal fluid (ILF) that contains these symbiotic bacteria. RNA from the ILF was extracted using the MasterPure™ RNA Purification Kit. After mRNA enrichment, an Illumina cDNA library was constructed. Approximatley 81% of the reads mapped to A. veronii or Rikenella. The Rikenella-like bacterium’s transcriptome indicated that energy is obtained by fermenting sugars to acetate. Surprisingly, the Rikenella-like symbiont foraged host mucin glycans rather than the blood meal. Its capacity to use mucins may be important for its ability to survive within the leech gut for up to 6 months between feedings. The discovery of mucin as the primary nutrient enabled the design of a medium that allowed the cultivation of Rikenella under conditions that favored its growth, and pure cultures were obtained. Thus, the application of metatranscriptomics to growing bacteria in diverse habitats can enable the design of media allowing their cultivation.

Bomar, L, et al. (2011). Directed culturing of microorganisms using metatranscriptomics. mBio, 2 (2) PMID: 21467263

RNA-Seq using Ribo-Zero-treated RNA aids study of chromatin-associated RNA

2011-10-21T10:33:00.000-07:00

The structural organization of actively transcribed regions of chromatin (euchromatin) is an area of intense interest. A recent report by Caudron-Herger et al. identified a new class of RNA that is associated with transcriptionally active chromatin. The authors termed these transcripts “chromatin-interlinking” RNAs or ciRNAs.

By combining fluorescence microscopy and microinjection of substrate-specific RNases, such as RNase A, RNase III, and RNase H, the authors conclude that ciRNA is single-stranded. They further characterized ciRNA by RNA-Seq using purified nuclear RNA fractions treated with the Ribo-Zero™ rRNA Removal Kit. ciRNA was found to compromise long (>500 nt) RNA polymerase II transcripts that were “spliced, depleted of polyadenylation and enriched with long 3'-untranslated regions (3’-UTRs) above ~800 nt."

Based on their results, the authors conclude that ciRNA “plays an important role in maintaining a decondensed and biologically active inter-phase chromatin conformation in human and mouse cell lines” and propose that ciRNA “could act as genome-organizing architectural factors of actively transcribed chromatin compartments.”

Caudron-Herger M et al. (2011). Coding RNAs with a non-coding function: Maintenance of open chromatin structure. Nucleus (Austin, Tex.), 2 (5) PMID: 21983088

Illumina launches Nextera DNA Sample Prep Kit

2011-10-11T11:22:00.000-07:00

Illumina today announced the availability of the Nextera™ DNA Sample Prep Kit. The new kit offers several advantages over the current Epicentre Nextera kits:

Fastest time to results: Go from DNA to data in less than 8 hours with Illumina’s MiSeq™ system.
Highest throughput: Index up to 96 samples and use master-mixed reagents to manually process > 500 samples per week.
Optimized PCR: Improved formulations with fewer cycles deliver reduced GC bias and lower error rates; PCR reagents are now bundled into reagent kits.
Improved insert size distribution: Support long paired-end 2x150 reads on MiSeq.
Increased process efficiency: Automation-friendly plate-based protocol.

Epicentre's Ilumina-compatible Nextera kits will be discontinued effective December 31, 2011.

Update: The Nextera DNA Sample Prep Kits (Roche 454-Compatible) will continue to be available for sale through Epicentre Customer Service until May 31, 2012, with delivery of final shipments required by June 30, 2012. We apologize for any inconvenience and hope this transition period will allow you to complete any ongoing projects.

Preparing high-quality DNA and RNA for Illumina sequencing

2011-09-27T14:06:00.000-07:00

The MasterPure™ line of DNA and RNA purification products has become one of the tools of choice for researchers wishing to obtain pure, high-quality nucleic acids for deep sequencing and microarray-based analysis. The MasterPure procedure uses gentle detergent/salt and proteolysis-based extraction technology that does not require the use of toxic organics or spin-columns. The resulting high-quality nucleic acids--RNA (RIN > 9.5) and DNA (A_260/280 >1.8)--are of high molecular weight. Recent publications have highlighted the use of MasterPure kits to generate DNA for standard Illumina^® library construction and GAII sequencing for developing reference genomes and de novo deep sequencing of various genomes. Other successful applications using MasterPure-purified DNA and RNA include SNP detection, methylation analysis, and expression profiling.

Platform	MasterPure™ Kit	Sample Type	Reference
GAII	MasterPure Gram-Positive DNA Kit	Bacteria (Riemerella anatipestifer type)	Mavromatis, K et al. (2011) doi:10.4056/sigs.1553862
	MasterPure Gram-Positive DNA Kit	Bacteria (Bacteroides salanitronis)	Gronow S et al. (2011) doi:10.4056/sigs.1704212
	MasterPure Complete Kit	Bacteria (Geobacter sulfurreducens)	Nagarajan H et al. (2010) doi:10.1371/journal.pone.0010922
	MasterPure Gram-Positive Kit	Bacteria (Aminomonas paucivorans)	Pitluck S et al. (2010) doi:10.4056/sigs.1253298
	MasterPure Gram-Positive DNA Kit	Bacteria (Marivirga tractuosa)	Pagani, I et al. (2011) doi:10.4056/sigs.1623941
GoldenGate™	MasterPure DNA Purification Kit	Colorectal carcinoma lines	Irizarry, RA et al. (2008) doi: 10.1101/gr.7301508
BeadArray™	MasterPure DNA Purification Kit	Blood lymphocytes; cystic fibrosis patients	Darrah R et al. (2010) doi: 10.1152/physiolgenomics.00185.2009

Nextera libraries aid in study of honey bee pathogens

2011-09-14T13:21:00.001-07:00

Recently, honey bee (Apis mellifera) populations in North America and in Europe have been experiencing increased annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Population loss of honey bee colonies poses grave risks to agriculture, due to the importance of these insects in pollination of food crops. Since the effect of environmental pathogens on bees has been poorly studied, the causes of CCD have not beeen well characterized. Thus, it is necessary to build a database of bee pathogens to learn more about pathogen transmission and to determine their role in CCD.

To this end, Runckel et al. wanted to identify what constitutes an abnormal pathophysiological condition in a honey bee colony. Using the Nextera™ DNA Sample Prep Kit (Illumina^®-compatible), the researchers developed pools of sequence data from 20 different monitor hives in an ultra-deep sequencing experiment, and these data were compared to sequence data from known bee pathogen types, including known viruses, Nosema sp., Crithidia mellificae, and bacteria. The authors state that theirs is the first U.S. honey bee pathogen monitoring study to report both comprehensive pathogen incidence and relative abundance of specific pathogens over time. Results from their molecular analysis pipeline (microarray, PCR, qPCR, ultra-deep sequencing) identified four novel RNA viruses, and provide a basis for future epidemiologic studies aimed at determining the causes of CCD.

Runckel, C. et al. (2011). Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia PLoS ONE, 6 (6) DOI: 10.1371/journal.pone.0020656

Nextera library prep used to characterize novel virus variants

2011-09-02T09:07:00.000-07:00

A significant advantage of Nextera™ library preparation technology is its low input requirements. A recent publication exploits this advantage to characterize the emergence and spread of a strain of new hemorrhagic fever viruses in red colobus monkeys in Uganda. Lauck et al. report that simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but has not been studied in wild primates. They describe the discovery and genetic characterization of two novel, divergent SHFV variants coinfecting a single male red colobus monkey found in the wild.

The genomic sequencing libraries were created using the Nextera DNA Sample Prep Kit (Roche Titanium-compatible) to locate and characterize any viral entities that may be residing in the wild. The Nextera libraries did not require isolation of viroids from the individual and only a simple total genomic DNA preparation was needed to isolate sufficient DNA (50 ng) for library construction and sequencing. Analysis of the sequence data revealed that the two viral genotypes seen show greater evolutionary sequence divergence than has been observed in other coinfecting virions that infect individuals within a population. The authors state that the results of this investigation will increase the knowledge base of the history and biology of these pathogens, and demonstrate that wild primates are often reservoirs for novel pathogens.

Lauck, M. et al. (2011). Novel, Divergent Simian Hemorrhagic Fever Viruses in a Wild Ugandan Red Colobus Monkey Discovered Using Direct Pyrosequencing PLoS ONE, 6 (4) DOI: 10.1371/journal.pone.0019056

Nextera technology enables complete sequencing of the CHO cell genome

2011-08-18T13:00:00.000-07:00

Chinese hamster ovary (CHO) cells are a workhorse in the production of therapeutic proteins. In a recent publication, Xu et al. report the complete sequencing of the CHO-K1 genome. Using the Nextera™ DNA Sample Prep Kit and the Illumina^® HiSeq 2000 sequencer, the researchers at BGI-Shenzen and other institutions generated a draft sequence of approximately 2.45 Gb with 24,383 predicted genes. They report these results as a means of studying glycosylation and viral susceptibility, in an attempt to understand the divergence of genomic sequence information. Even among clones of CHO cells containing engineered or other therapeutic proteins, DNA translocations/rearrangements can occur. Prior to generating this draft sequence, the main tool for analyzing genome-scale changes has largely been limited to the use of expressed sequence tags (ESTs). The study describes how the availability of the CHO genome assists in understanding how protein glycosylation and viral susceptibility affect yields and the quality of production of therapeutic proteins. Based on these insights, the researchers expect enhanced application of CHO cell engineering for protein manufacturing.

Xu, X. et al. (2011). The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line Nature Biotechnology, 29 (8), 735-741 DOI: 10.1038/nbt.1932

Fosmid libraries aid in identifying a novel lipase

2011-08-04T13:37:00.000-07:00

The CopyControl™ Fosmid Library Production Kit remains a key tool in the discovery and sourcing of novel, useful enzymes. In a recent publication, Glogauer et al. identified a novel lipase gene from a fosmid metagenomic library constructed with "prokaryotic-enriched" DNA from fat-contaminated soil collected from a wastewater treatment plant. The library, containing over 500,000 unique clones, revealed esterase activity in 32 of the library member clones after screening on agar plates containing 1% tricaprylin, or 1% triolein (denoting the presence of true lipases). One of these clones produced a novel lipase enzyme that the authors believe will be a useful tool in biocatalytic processes.

The researchers state that the novel lipase possesses “high specific activity against long-chain triacylglycerols, activity and stability over a wide range of pH values, good thermal stability, and stability in water-miscible organic solvents and at high salt concentrations.”

Thus, even with the growth of high-throughput sequencing technologies, CopyControl fosmid libraries continue to assist in the discovery of novel activities in metagenomic populations that may not be possible in high-copy-number cloning procedures.

Glogauer, A. et al. (2011). Identification and characterization of a new true lipase isolated through metagenomic approach Microbial Cell Factories, 10 (1) DOI: 10.1186/1475-2859-10-54

Species compatibility for Ribo-Zero rRNA Removal Kits

2011-07-27T12:27:00.000-07:00

Ribo-Zero™ rRNA Removal Kits are becoming the method of choice for RNA-Seq workflows, especially for degraded or compromised RNA samples. We often get questions from customers who want to know whether we have a Ribo-Zero kit for a particular organism. The information below offers some guidance on selecting the appropriate kit.

If you've used a Ribo-Zero kit for a species not listed here, e-mail your results to techhelp@epicentre.com, and we''ll send you a free Epicentre 2 GB flash drive!

Species	Ribo-Zero Kit (Human/Mouse/Rat) Compatibility
Human	>99% removal of 28S, 18S, 5.8S and 5S rRNAs
Mouse	>99% removal of 28S, 18S, 5.8S and 5S rRNAs
Rat	>99% removal of 28S, 18S, 5.8S and 5S rRNAs
Other mammalian (e.g., cow, pig)	Not tested but expected to perform well.
Drosophila	Will not remove 5S rRNA or 'right' fragment of 28S rRNA.
C. elegans	Use highest quality RNA possible.
Yeast	Removes 26S, 18S, 5.8S rRNAs from intact sample. Will not remove 5S rRNA. Use highest quality RNA possible.
Xenopus	Use highest quality RNA possible.
Mosquito	Not recommended
Zebra fish	Efficient removal of 28S, 18S, and 5S rRNAs. 5.8S rRNA removal has not been tested.
Lamprey	Use highest quality RNA possible.
Shrimp	Not recommended
Aspergillus niger	300 nt region of 18S rRNA will not be removed from a degraded sample. Use highest quality RNA possible.

Species	Ribo-Zero Kit (Gram-Negative Bacteria) Compatibility
E. coli	>99.9% removal of 23S, 16S and 5S rRNAs
Lactobacillus plantarum	>99% removal of 23S, 16S and 5S rRNAs
Salmonella typhimurium	Efficiently removes 16S and 5S rRNAs. Does not remove a 107 nt region of the fragmented 23S rRNA.
Campylobacter	Efficiently removes 23S and 16S rRNAs. Will not remove 5S rRNA.
Pseudomonas aeruginosa	Efficiently removes 23S, 16S and 5S rRNAs.
Porphyromonas gingivalis	Expected to efficiently remove 23S, 16S rRNAs. May not remove 5S rRNA.
Cyanobacteria	Efficiently removes 23S, 16S and 5S rRNAs.
Photorhabdus	Efficiently removes 23S, 16S and 5S rRNAs.
Francisella	Expected to efficiently remove 23S, 16S, 5S rRNAs.

Species	Ribo-Zero Kit (Gram-Positive Bacteria) Compatibility
Bacillus subtilis	Removes >99% of 23S, 16S and 5S rRNAs.
Mycobacterium tuberculosis	Efficiently removes 23S, 16S and 5S rRNAs.
Streptococcus	Expected to efficiently remove 23S, 16S, 5S rRNAs.
Clostridium difficile	Expected to efficiently remove 23S, 16S, 5S rRNAs.

EZ-Tn5 Custom Transposon Contruction Kits now available

2011-07-08T12:31:00.000-07:00

Epicentre's new EZ-Tn5™ Custom Transposome Construction Kits, with a choice of two different EZ-Tn5 Transposon Construction Vectors, are designed for in vitro and in vivo use. Simply construct the transposon using nearly any DNA in the included pMOD-2 or pMOD-3 Transposon Construction Vector and purify the transposon. The transposon can then be used in vitro to transpose or mutagenize any target DNA. For in vivo transposon mutagenesis, form a complex with the included EZ-Tn5 Transposase in a simple, 30-minute reaction, and electroporate into the microbe of interest. EZ-Tn5 Transposomes do not require suicide vectors or Tn5 transposase expression cassettes--simply electroporate and screen. EZ-Tn5 Transposomes have been used in a wide variety of organisms.

Something old, something new: Nextera and CopyControl technologies

2011-06-29T14:25:00.000-07:00

With the advent of next-generation sequencing techniques, genomic cloning has taken on a new role in de novo and reference-genome sequencing. Previously, Kitzman et al.¹ generated massive amounts of shotgun sequence data from a human library by sequencing 120 pools of CopyControl™ fosmid clones using Nextera™ DNA library preparation technology. This method generated sequencing data of sufficient depth to assemble a complete haplotype-resolved genome for the individual being studied.

However, a recent study by Naka et al.² used a combination of direct genomic next-generation sequencing and CopyControl fosmid cloning to generate and confirm the de novo sequence of the bacterium Vibrio anguillarum strain 775. While deep sequencing generated 32X coverage of the bacterial genome, there were gaps in the sequence data that required confirmation, or contig “gap-filling.” The CopyControl fosmid library allowed the generation of sequence data confirming not only the taxonomy of the new organism, but also demonstrating the synergistic approach of using deep sequencing techniques together with classical Sanger-based sequencing.

1. Kitzman, J. et al. (2010). Haplotype-resolved genome sequencing of a Gujarati Indian individual Nature Biotechnology, 29 (1), 59-63 DOI: 10.1038/nbt.1740
2. Naka, H. et al. (2011). Complete Genome Sequence of the Marine Fish Pathogen Vibrio anguillarum Harboring the pJM1 Virulence Plasmid and Genomic Comparison with Other Virulent Strains of V. anguillarum and V. ordalii Infection and Immunity, 79 (7), 2889-2900 DOI: 10.1128/IAI.05138-11

Summer stock-up special

2011-06-23T07:10:00.000-07:00

Epicentre would like to help make your summer even better this year. That's why we're offering iTunes® Gift Cards to customers who want to stock up on Epicentre products:

Purchase $2,500 to $4,999 of Epicentre products on a single invoice and receive a free $25 iTunes Gift Card.
Purchase $5,000 or more of Epicentre products on a single invoice and receive a free $50 iTunes Gift Card.

You can use your gift card for music, movies, TV shows, books, and apps for your iPhone®, iPod®, or iPad®—anything that's available in the Apple® iTunes Store.

For more information, see: www.epicentre.com/itunes

Nextera technology featured at ASM 111th General Meeting

2011-06-06T13:37:00.000-07:00

Less is more--Evaluation of a low-input, transposase-mediated protocol for rapid generation of high-throughput sequence libraries

Epicentre’s Nextera™ library preparation kits have taken the metagenomics field by storm. Researchers are taking advantage of the rapid, simple Nextera library preparation to determine species, as well as specific enzyme activities, present in a given environmental sample. In a poster at the 111th General Meeting of the American Society for Microbiology (ASM) in New Orleans, Marine et al. used the Nextera DNA Sample Prep Kit (Roche 454-Compatible) to dissect a mock sample in a cultivation-independent bacteriophage preparation. The library, consisting of five mycobacteriophages and four cyanophages, had a relatively broad range of GC content (35.3%-64.7%) and read abundance (28.1%-0.3%).

The results showed that the Nextera library preparation did not compromise the relative abundance of each of the phages in the preparation, and scarce members of the population remained detectable in the presence of more populous library members. The assembled genomes of four of the phages from the Nextera-generated sequence data covered >99% of the genomes at near-perfect identity. GC content was found to affect the coverage of the genomes, though the researchers attributed part of the GC bias to amplification bias in the emulsion PCR prior to sequencing.

The researchers concluded that the Nextera technology is a good alternative to older library preparation methods, and generated quality sequence data from a variety of origins from small quantities (50 ng) of input DNA.

Visit Epicentre at the ASM 2011 General Meeting

2011-05-13T12:05:00.000-07:00

Epicentre will be at the 111th American Society for Microbiology General Meeting, March 22-24, 2011, at the New Orleans Convention Center. Stop by booth 1737 to learn about our novel Nextera™ library preparation products for next-generation sequencing, ScriptSeq mRNA-Seq library preparation products, and Ribo-Zero™ rRNA removal kits.

We’ll be available to guide you through the best solutions for your research, including our broad assortment of kits for nucleic acid purification, PCR, genomic library production, and the EZ-Tn5™ Transposon tools. Exhibit hours are Sunday and Monday, May 22 and 23 from 10:30 a.m. through 4 p.m., and Tuesday, May 24, from 10:30 a.m. through 2:30 p.m.

We look forward to meeting you at the conference!

Ribo-Zero rRNA removal from FFPE RNA

2011-05-10T14:41:00.000-07:00

One of the key benefits of the Ribo-Zero™ family of rRNA removal products is their ability to remove rRNA from partially degraded total RNA samples. We recently presented data on the performance of Ribo-Zero Kits with formalin-fixed, paraffin-embedded (FFPE) samples at the AACR 2011 Annual Meeting.

RNA from FFPE breast tumor tissue was used as starting material to prepare a mRNA-Seq library. The RNA sample was treated with the Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) prior to library preparation using the ScriptSeq™ mRNA-Seq Library Preparation Kit (Illumina^®-Compatible). Single-lane, 54-nt unidirectional sequencing reads were obtained using an Illumina^® GAII sequencer, and sequence alignment was performed by Beijing Genomics Institute (BGI) with both the well-annotated human genome (ENSEMBL56) and in-house built junction sequences using BWA software allowing for four mismatches. The results were compared to those obtained from a library prepared without Ribo-Zero treatment.

(click to enlarge figure)

The Ribo-Zero-treated sample contained 76% of all 49,222 possible expressed genes with 0.24% of the reads being rRNA, compared to 48.8% rRNA reads for the untreated samples. As seen in the figure, Ribo-Zero treatment significantly increases mapped reads for exon, intergenic, and intron transcripts.

Nextera library primers demystified

2011-04-01T12:37:00.000-07:00

We get a number of inquiries about whether the Nextera™ Read 1, Read 2, and Index Read sequencing primers are compatible with the standard Illumina® sequencing primers. The Nextera and Illumina sequencing primers are indeed compatible with each other. The sequences of Nextera primers are different from standard Illumina primers, which means that they will not interfere with or cross-hybridize to each other. Nextera primers also will not anneal to a library created using standard Illumina sample prep technology and vice versa. This means that it is possible to sequence both libraries, not only in the same flow cell, but also in the same channel, as long as the libraries are suitably barcoded.

For libraries prepared using Illumina TruSeq™ technology: as with the standard Illumina libraries, Nextera libraries can also be sequenced in the same channel of the same flow cell, again due to the differences between Nextera primer and TruSeq primer sequences. Thus, with appropriate barcoding, it is possible to sequence a Tru-Seq library, a standard Illumina-generated library, and a Nextera library in the same flow cell, without fear of primer interferences or sequence data confusion.

Preparing Nextera libraries from short linear DNA

2011-03-16T02:18:00.000-07:00

Occasionally, we get requests for information on the use of the Nextera™ DNA Sample Prep products for sequencing short linear DNA, such as PCR products or cDNA libraries. Nextera kits can be used with these targets, though a minor change in the procedure will be required.

Due to the mechanism of DNA “tagmentation” by Nextera Transposomes, the end sequences (the last 100-150 bases) of the target DNA will have lower depth of coverage than the center of the DNA. This is because the Transposomes are not capable of adding adaptors to the ends of the DNA, and because at least two Nextera transposon insertions are required to tagment a given DNA molecule. A relatively simple fix is to design PCR or cDNA primers that include one of the Nextera Adaptor 1 and Adaptor 2 sequences; alternatively, ligate the adaptor sequences to the ends of the amplicon or ds cDNA. This will allow the end sequences to participate in the limited-cycle PCR after tagmentation, and allow for improved coverage of the ends of the DNA.

Another option when using PCR amplicons is to design PCR primers so that the priming sites are outside the desired region for sequencing. This will increase the likelihood that a Nextera Transposome complex will insert much closer or next to the end of the sequence being studied.

Visit Epicentre at the CHI XGen Congress

2011-03-09T13:48:00.000-08:00

Epicentre will be attending the CHI XGen Congress, March 14-18, 2011, in San Diego, CA. Stop by booth #23 to learn more about speeding up DNA and RNA sample prep for next-generation sequencing. We’ll also have special offers and discounts on select Epicentre products, available exclusively to XGen Congress attendees.

To learn more about Nextera™ technology, don’t miss the following presentations:

Thursday, March 17, 7:45 a.m.
Breakfast presentation: Eliminating the Library Preparation Bottleneck, by Nick Caruccio, Ph.D. (Epicentre Biotechnologies)

Thursday, March 17, 2:40 p.m.
Rapid Construction of Complex, Low-Input, Low-Bias Fragment Libraries for Massively Parallel DNA Sequencing by Transposase-Catalyzed Adaptor Insertion, by Andrew Adey (University of Washington)

Also, visit our posters:

Nextera™ PCR-Free DNA Library Preparation for Next-Generation Sequencing
Novel Technologies for Ribosomal RNA Removal and Directional RNA-Seq Library Preparation

If you’re not able to attend the meeting and would like more information on our featured products, please contact us by e-mail or call 1 (800) 284-8474 within the US.

Circligase II-based detection of miRNAs

2011-02-10T11:21:00.000-08:00

In a recent publication, Kumar et al. demonstrate the power of their “miR-ID” method in expression profiling of microRNA (miRNA) molecules. The authors used a ligase-based recircularization procedure and compared T4 RNA Ligase to Epicentre’s Circligase™ II enzyme (typically used for ssDNA circularization) in recircularization of miRNAs. They found that, while both ligases were able to join the ends of standard miRNA molecules with 2’ and 3’-hydroxyl groups, only Circligase II-based reactions were able to ligate the ends of miRNAs that contained 2’-O-methyl groups (common in plant miRNAs). Following recircularization, the miRNAs were reverse-transcribed to form tandem repeats of the sequence that is complementary to the miRNA, and then amplified by qPCR to develop an expression profile of miRNAs in a given sample.

The authors state that no chemically modified probes or primers are required for their method. The key elements of the miR-ID process (miRNA circularization, reverse transcription of circularized RNA, and qPCR using 5’-overlapping primers) are likely to be of general interest in other transcriptome discovery techniques.

Kumar, P. et al. (2010). miR-ID: A novel, circularization-based platform for detection of microRNAs RNA, 17 (2), 365-380 DOI: 10.1261/rna.2490111

Nextera™ sample prep enables breakthrough study of copy-number variation

2011-01-18T09:31:00.000-08:00

The study of copy-number variation (CNV) in humans has contributed to our understanding of genetic uniqueness, as well as disease. Until recently, it was difficult to assess the number of repeated DNA sequences in the genome. In a recent publication, researchers at the 1000 Genomes Project and collaborators have invented new methods to study and find repetitive DNA sequences in the human genome, and have found that CNVs occur in only 7%-9% of human genes. They used the new techniques to compare the entire genomes of 159 individuals and were able to accurately assay previously unidentified duplicated genes.

For sequencing, the researchers picked and cultured 144 fosmid clones (from libraries prepared by shotgun cloning of genomic DNA) from eight selected individuals. After fosmid DNA purification, clone DNA was arrayed in a 96-well plate (two clones combined from unrelated loci for some). Bar-coded sequencing libraries were created separately from each well using the Nextera DNA Sample Prep Kit (Illumina-compatible), using 100 ng of fosmid DNA per well as the starting material. The 96 bar-coded Nextera libraries were pooled and sequenced on two lanes of an Illumina GAII (paired-end, 2 x 76-bp reads, with an additional 9-bp index read). Reads were mapped to the genome and analyzed as described in the supplementary information.

The authors report:

"We identified 4.1 million ‘singly unique nucleotide’ positions informative in distinguishing specific copies…these data identify human-specific expansions in genes associated with brain development, reveal extensive population genetic diversity, and detect signatures consistent with gene conversion in the human species. Our approach makes ~1000 genes accessible to genetic studies of disease association."

Sudmant, P. et al. (2010). Diversity of Human Copy Number Variation and Multicopy Genes Science, 330 (6004), 641-646 DOI: 10.1126/science.1197005

Illumina acquires Epicentre Biotechnologies

2011-01-12T08:11:00.000-08:00

Illumina Acquires Epicentre Biotechnologies, Leading Provider of Nucleic Acid Sample Preparation Reagents and Specialty Enzymes
Combination Enhances Illumina's Sample Preparation and Enzyme Portfolio

SAN DIEGO, Jan 11, 2011 (BUSINESS WIRE) -- Illumina, Inc. (NASDAQ:ILMN) today announced that it has acquired Epicentre Biotechnologies, a leading provider of nucleic acid sample preparation reagents and specialty enzymes used in sequencing and microarray applications. A key component of the acquisition is direct access to Epicentre's proprietary Nextera(TM) technology for next-generation sequencing library preparation, which greatly simplifies genetic analysis workflows and reduces time from sample preparation to answer.

"As next-generation sequencing continues to improve in throughput and cost, there's a critical need for sample prep to evolve as well, to lower costs, handle higher sample volumes and reduce both hands-on and overall processing time," said Jay Flatley, President and CEO of Illumina. "Epicentre's Nextera technology provides a step-change improvement in library prep that will translate into greater ease of use, lower costs, and faster turnaround times for sequencing applications. In addition to Nextera, Epicentre is a leading supplier of specialty enzymes and kits that are beneficial to Illumina's technologies."

The rapid adoption of Nextera sequencing sample prep kits by existing Illumina sequencing customers is indicative of the cost effectiveness, ease of use, and efficiency of Nextera technology. With this patented technology, researchers can prepare sequencer-ready libraries from genomic DNA with less than 15 minutes of hands-on time - a significant timesaving compared to alternate methods. In addition, Nextera technology requires 10-100 times less starting DNA, which enables applications with limited starting material such as tumor biopsies, degraded DNA, or purified RNA. These unique features of Nextera sequencing library prep kits are all critical to advancing the evolution of next-generation sequencing.

The combined company will be uniquely positioned to offer an end-to-end solution for next-generation sequencing, microarray, and real time PCR applications. Epicentre's unique capabilities in enzyme engineering and sample preparation reagent development will complement Illumina's core platform expertise to comprehensively address the needs of researchers across their entire genetic analysis workflow.

About Illumina

Illumina (www.illumina.com) is a leading developer, manufacturer, and marketer of life science tools and integrated systems for large-scale analysis of genetic variation and function. We provide innovative sequencing and array-based solutions for genotyping, copy number variation analysis, methylation studies, gene expression profiling, and low-multiplex analysis of DNA, RNA, and protein. We also provide tools and services that are fueling advances in consumer genomics and diagnostics. Our technology and products accelerate genetic analysis research and its application, paving the way for molecular medicine and ultimately transforming healthcare.

Forward-Looking Statements

This release contains forward-looking statements that involve risks and uncertainties. Important factors that could cause actual results to differ materially from those in any forward-looking statements include challenges inherent in integrating Epicentre with our existing operations and the other factors that are detailed in our filings with the Securities and Exchange Commission, including our most recent filings on Forms 10-K and 10-Q, or in information disclosed in public conference calls, the date and time of which are released beforehand. We do not intend to update any forward-looking statements after the date of this release.

SOURCE: Illumina, Inc.

Illumina, Inc.
Investors:
Peter J. Fromen
Sr. Director, Investor Relations
858-202-4507
pfromen@illumina.com
or
Media:
Wilson Grabill
Sr. Manager, Public Relations
858-882-6822
wgrabill@illumina.com

Visit Epicentre at the PAG XIX Conference

2011-01-10T12:29:00.000-08:00

Epicentre will be attending the International Plant and Animal Genome (PAG) XIX Conference, to be held from January 15-19 in San Diego, CA. Stop by Booth #331 to learn more about our products for DNA- and RNA-Seq library preparation, and to receive special discounts on a variety of Epicentre products.

In addition, we will be presenting the following posters:

Nextera™ Technology for Directional and Nondirectional RNA-Seq Expression Analysis on the Illumina Platform
Improved Technologies for Ribosomal RNA Removal and Directional RNA-Seq Library Preparation
Enhanced Methods To Capture the Entire Small-RNA Transcriptome for RNA-Seq

Enhanced mapping of the C. elegans transcriptome through multiple RNA-Seq techniques

2011-01-06T14:12:00.000-08:00

In a recent publication, Lamm et al. demonstrated the use of Epicentre’s CircLigase ssDNA Ligase for template generation in high-throughput RNA-Seq applications. The authors used a combination of three high-throughput RNA capture and sequencing methods to refine and augment the transcriptome map of Caenorhabditis elegans. The CircLigase method relies on RNA fragmentation, poly(A) tailing, and oligo(dT)-based hybrid capture to generate a cDNA that can be recircularized and PCR-amplified for high-throughput sequencing. The other two methods involved single-strand RNA ligation, and double-stranded cDNA linker ligation.

The authors found that each RNA-Seq approach shows specific limitations and biases, and the use of multiple methods provided a more complete map than was obtained from any single method. They also note the advantages of CircLigase-based and ssRNA-based capture for locating and sequencing the exact 5’ ends of the RNAs, which were extremely difficult to capture using the double-stranded cDNA capture method.

Lamm, A. et al. (2010). Multimodal RNA-seq using single-strand, double-strand, and CircLigase-based capture yields a refined and extended description of the C. elegans transcriptome Genome Research DOI: 10.1101/gr.108845.110

Comparison of various DNA-Seq library prep methods

2010-12-23T12:11:00.000-08:00

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.

Adey, A. et al. (2010). Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition Genome Biology, 11 (12) DOI: 10.1186/gb-2010-11-12-r119

Quantitation of input DNA and Nextera™ fragment size distribution

2010-12-20T14:43:00.000-08:00

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.

BAC libraries enable phylogenetic analysis in Japanese pear

2010-12-15T09:08:00.000-08:00

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.

Okada, 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

Microsatellite loci for Symbiodinium A3 Identified using next-generation sequencing

2010-12-10T13:34:00.000-08:00

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.

Pinzón, J. et al. (2010). Microsatellite loci for Symbiodinium A3 (S. fitti) a common algal symbiont among Caribbean Acropora (stony corals) and Indo-Pacific giant clams (Tridacna) Conservation Genetics Resources, 3 (1), 45-47 DOI: 10.1007/s12686-010-9283-5

New small-RNA-Seq library prep kit

2010-12-02T13:29:00.000-08:00

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.

New kits for rRNA removal

2010-11-12T13:29:00.000-08:00

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.

rRNA removal from Drosophila total RNA

2010-10-27T13:41:00.000-07:00

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.

Obtaining optimal RNA-Seq library quality

2010-10-13T14:46:00.000-07:00

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).

Examining blocking lesions in ancient DNA

2010-10-07T13:29:00.000-07:00

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.

Heyn, 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

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

2010-09-30T12:27:00.000-07:00

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.”

Bimber, B. et al. (2010). Whole genome characterization of HIV/SIV intra-host diversity by ultra-deep pyrosequencing Journal of Virology DOI: 10.1128/JVI.01378-10

Share your research results with EpiCentral readers!

2010-09-24T07:22:00.000-07:00

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.

Posts should be approximately 500 words, with up to two figures or tables.
Text may be submitted as Microsoft Word (.doc or .docx), RTF, or plain-text files. Images should be submitted as JPEG or TIFF files.
Submit all materials by e-mail to: forum@epibio.com
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.

Nextera libraries from buccal-cell DNA

2010-09-17T13:30:00.000-07:00

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.

Centrifuge the tube briefly (1 minute at 3,000 x g) to remove the solid cellular debris.
Remove the supernatant and precipitate the DNA with 1/10 volume of sodium acetate (3.0 M) and 2 volumes of ethanol.
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).
Add 300 µl of Binding Buffer and follow the manufacturer’s protocol.
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.

Size distribution of Nextera Illumina-compatible libraries

2010-09-09T12:52:00.000-07:00

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

HPV prevalence in esophageal squamous cell carcinoma

2010-09-02T13:32:00.000-07:00

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.

Antonsson, A. et al. (2010). High-Risk Human Papillomavirus in Esophageal Squamous Cell Carcinoma Cancer Epidemiol Biomarkers Prev, 19 (8), 2080-2087 DOI: 10.1158/1055-9965.EPI-10-0033

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

2010-08-26T14:44:00.000-07:00

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.

Li, 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

Using Nextera technology for RNA-Seq expression analysis

2010-08-19T13:04:00.000-07:00

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

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

2010-08-16T13:01:00.000-07:00

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:

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”.

Add 250 µl of isopropanol to both tubes, invert 30-40 times to mix.
Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
Rinse with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove residual ethanol with a pipet.

DNA Protocol (for the tube marked “DNA”)

Resuspend the pellet in 100 µl TE Buffer.
Add 1 µl of RNase A to sample and mix well.
Incubate @ 37°C for 10 minutes. Note: Additional incubation (up to 30 minutes) may be needed.
Add 100 µl of 2X T&C Lysis Solution, and mix by vortexing for 5 seconds.
Add 100 µl of MPC protein precipitation reagent, mix by vortexing for 10 seconds. Place on ice for 3-5 minutes.
Centrifuge for 10 minutes at ≥10,000 x g.
Transfer the supernatant to a new microcentrifuge tube and discard the pellet.
Add 250 µl of isopropanol. Invert 30-40 times to mix.
Centrifuge the tubes for 10 minutes at maximum speed in a microcentrifuge at 4°C.
Carefully remove the isopropanol with a pipet, taking care not to dislodge the pellet.
Rinse twice with 70% ethanol. Centrifuge briefly if the pellet is dislodged. Remove all residual ethanol with a pipet.
Resuspend the DNA in 10-35 µl of TE Buffer.

RNA Protocol (for the tube marked “RNA”)

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.
Resuspend the pellet in 100 µl of DNase I solution.
Continue with steps 3 through 12 from the DNA Protocol (above).

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

2010-08-11T13:02:00.000-07:00

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

Fosmid cloning enables new techniques in synthetic biology

2010-08-05T14:00:00.000-07:00

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.

Sommer, 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

Solutions for problematic plant templates

2010-07-29T11:39:00.000-07:00

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

Fosmid cloning: Alive and kicking

2010-07-14T14:55:00.000-07:00

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.

1. 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

When is “direct PCR” not direct?

2010-07-09T12:17:00.000-07:00

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.

Gene expression analysis from Ribo-Zero™-treated RNA

2010-07-06T14:25:00.000-07:00

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.

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.)

Functional metagenomics reveals mechanisms of antibiotic resistance

2010-06-25T11:52:00.000-07:00

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.

Donato, J. et al. (2010). Metagenomic Analysis of Apple Orchard Soil Reveals Antibiotic Resistance Genes Encoding Predicted Bifunctional Proteins Applied and Environmental Microbiology, 76 (13), 4396-4401 DOI: 10.1128/AEM.01763-09

Superior ribosomal RNA (rRNA) removal for RNA-Seq

2010-06-22T13:51:00.000-07:00

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%

Get rid of the small stuff

2010-06-18T13:12:00.000-07:00

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).

Visit our poster at the CHI Beyond Sequencing meeting

2010-06-15T14:00:00.000-07:00

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.

Library preparation for ChIP-Seq

2010-06-08T15:28:00.000-07:00

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.

Cheung, I. et al. (2010). Developmental regulation and individual differences of neuronal H3K4me3 epigenomes in the prefrontal cortex Proceedings of the National Academy of Sciences, 107 (19), 8824-8829 DOI: 10.1073/pnas.1001702107

From cows to chickadees: BuccalAmp™ Kits in animal genomics

2010-06-04T02:31:00.000-07:00

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.

ASM 2010: A good week for Epicentre

2010-06-01T13:03:00.000-07:00

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).

Purifying bacterial DNA from contaminated food samples

2010-05-24T12:22:00.000-07:00

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.

Lambertz, S. et al. (2008). TaqMan-Based Real-Time PCR Method for Detection of Yersinia pseudotuberculosis in Food Applied and Environmental Microbiology, 74 (20), 6465-6469 DOI: 10.1128/AEM.01459-08

Structural differences between Marburg and Ebola viruses

2010-05-18T08:52:00.000-07:00

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.

Enterlein, S. et al. (2009). The Marburg Virus 3' Noncoding Region Structurally and Functionally Differs from That of Ebola Virus Journal of Virology, 83 (9), 4508-4519 DOI: 10.1128/JVI.02429-08

Visit Epicentre at ASM 2010 in San Diego, CA

2010-05-14T11:57:00.000-07:00

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.

Isolation of RNA from breast cancer FFPE tissues

2010-05-11T14:42:00.000-07:00

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.

Sánchez-Navarro, I. et al. (2010). Comparison of gene expression profiling by reverse-transcription quantitative PCR between fresh frozen and formalin-fixed, paraffin-embedded breast cancer tissues BioTechniques, 48 (May 2010), 389-397 : 10.2144/000113388

Nextera™ library fragment size range

2010-05-06T14:22:00.000-07:00

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.

Transposon mutagenesis identifies a novel toxin regulatory locus in Clostridium perfringens

2010-05-03T13:20:00.000-07:00

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.

*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

Identification of suitable hosts for small-molecule functional metagenomics

2010-04-29T11:09:00.000-07:00

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.

*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

Labeling RNA for gene expression analysis

2010-04-27T03:00:00.000-07:00

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.

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

2010-04-23T11:07:00.000-07:00

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.

*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

Complete Genomics sequencing platform built on Epicentre’s enzymes

2010-04-20T13:41:00.000-07:00

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:

PlasmidSafe™ ATP-Dependent DNase, to eliminate residual linear DNA after adaptor ligation and dsDNA circularization;
Exonuclease I and Exonuclease III to eliminate noncircularized DNA after the formation of ssDNA circular templates; and
T4 Polynucleotide Kinase to activate the 3’ and 5’ ends of anchors.

*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

FailSafe™ PCR System earns praise

2010-04-15T03:12:00.000-07:00

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.

CHI XGen Congress session on Nextera™ technology

2010-04-13T14:35:00.000-07:00

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.

Save big on library prep for Roche GS FLX

2010-04-09T07:52:00.000-07:00

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

Transposomics made EZ

2010-04-06T15:14:00.000-07:00

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.

Nextera™ tagmentation of soil metagenomic DNA

2010-03-30T11:12:00.000-07:00

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.

Nextera™ libraries and sequencing primers

2010-03-23T14:42:00.000-07:00

We are often asked about the transposon end sequence inserted during the Nextera™ library prep procedure (see technology overview), and how this sequence relates to those of the sequencing primers.

I’m running an Illumina sequencer and don’t want to read through the 19-bp transposon sequence. Isn’t this a waste of limited Illumina data?
The Nextera Illumina libraries are sequenced with a custom sequencing primer (provided in the kit). The first read is the genomic fragment. There are no wasted reads with the Illumina libraries.

Are you sure your Nextera-Illumina sequencing primers are compatible with the Illumina system?
Yes. The Nextera sequencing primers have been fully validated and are completely compatible with the standard Illumina sequencing primers. We have even sequenced a standard Illumina library and a Nextera library in the same channel of the same flow cell. The primers do not interfere with each other.

Do you read through the 19-bp transposon sequence on the Roche/454 sequencing platform?
Yes. Nextera libraries are sequenced using the standard Roche/454 primers. As a result, the first reads are the QC key, followed by any added bar coding, followed by the 19-bp transposon sequence. The transposon sequence must be filtered/masked prior to mapping and assembly.
Transposon Sequence: 5'-AGATGTGTATAAGAGACAG-3'

Terminator™ Exonuclease helps unravel the mysteries of the Helicobacter pylori transcriptome

2010-03-18T14:02:00.000-07:00

Helicobacter pylori infects about 50% of the human population, and is implicated in inflammation, ulcers, and gastric cancer. Sharma et al.* used a novel approach--differential RNA-Seq (dRNA-Seq)--that selects for the 5’ ends of primary transcripts to construct a genome-wide map of transcriptional start sites (TSS) and operons. Their analysis revealed the unexpected complexity of the H. pylori transcriptome, and demonstrated that the dRNA-Seq method has wide-ranging potential for studying gene expression in pathogenic organisms.

A key component of the dRNA-Seq method is the discrimination of primary transcripts (with 5’-triphosphate ends) from processed ones (with 5’-monophosphate ends). The authors used Terminator™ Exonuclease to enrich a total bacterial RNA preparation in primary transcripts. Terminator Exonuclease degrades transcripts that have a 5’-monophosphate end, but not those that have a 5’-triphosphate, 5’-capped, or 5’-hydroxyl end. The authors conclude that:

Other RNA-seq studies detected termini of bacterial transcripts but could not unequivocally assign TSS due to lack of 5’ group discrimination. As 5’-[triphosphate] ends mark native transcripts in all eubacteria, dRNA-seq should help improve the genome annotations of other organisms, alone or through metatranscriptomics.

*Sharma, C. et al. (2010). The primary transcriptome of the major human pathogen Helicobacter pylori Nature, 464 (7286), 250-255 DOI: 10.1038/nature08756

Lower prices, larger sizes for Nextera™ kits

2010-03-15T05:00:00.000-07:00

Effective today, Epicentre is lowering prices on all Nextera™ DNA Sample Prep Kits. In addition, kits are now available in 50- and 100-reaction sizes for Roche Titanium-compatible and Illumina-compatible platforms. These changes were made as a direct result of your feedback, so please continue to offer your comments and suggestions. As before, we will offer special pricing on bulk orders (i.e., larger than 100 reactions).

Nextera™ technology at the CHI XGen Congress

2010-03-10T07:51:00.000-08:00

Epicentre representatives will be showcasing Nextera™ DNA Sample Prep technology at the CHI XGen Congress, March 15-19, 2010 in San Diego, CA.

Breakfast Presentation: Wednesday March 17, 2010, 7:30 AM
Nextera™ Library Preparation: From Nanograms of DNA to Sequencer-Ready Libraries in Less Than Two Hours
by Nicholas Caruccio, PhD

“Java and Jive” Discussion Group: Wednesday March 17, 2010, 8:15 AM
Table 4: Advances in NGS Library Preparation
Host: Nicholas Caruccio, PhD
Discussion will be focused on:

Limiting and challenging samples
Simplified workflow
High-throughput methods
Assessing library quantity and quality

In addition, Epicentre will be presenting a poster: Simplified DNA Library Preparation: Simultaneous DNA Fragmentation and Adaptor Tagging by In Vitro Transposition.

Extracting DNA or RNA from FFPE Tissues

2010-03-08T14:56:00.000-08:00

BioCompare recently published an overview of the challenges associated with extracting DNA or RNA from formalin-fixed, paraffin-embedded (FFPE) tissues. The article looks at various commercially available options for extracting nucleic acids from FFPE samples, including Epicentre’s QuickExtract™ FFPE DNA and RNA kits, and the MasterPure™ Kits. The article cites a review of the QuickExtract FFPE DNA Extraction Kit published on BioCompare by Dr. Michael Campa, Duke University, in which he states:

I was attracted to this kit because the protocol required no deparaffinization of the specimen prior to DNA extraction. Deparaffinization is usually accomplished with 2 to 3 incubations in xylene and can be a nuisance, particularly if you have more than just a few specimens to deal with.

Dr. Campa found that all 40 samples he processed using the kit gave him strong bands in PCR, and the gel-purified amplicons provided excellent sequencing results.

RNA can prove even more challenging to extract from FFPE samples than DNA, due to the greater extent of degradation over time. Glenn et al.* compared the performance of RNA isolated from FFPE tissue slices in TaqMan® qPCR assays. They concluded that:

When using FFPE tissue, the MasterPure kit produced the highest RNA yield with no contaminating genomic DNA. RNA yield can be further enhanced by including an overnight Proteinase K digestion. Gene-specific primers enhance reverse transcription and increase the sensitivity of qPCR.

* Glenn, S., Head, K., Teh, B., Gross, K., & Kim, H. (2009). Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis Journal of Biomolecular Screening, 15 (1), 80-85 DOI: 10.1177/1087057109355059

ChIP-Seq used to examine diverse roles of transcription factors

2010-03-04T13:15:00.000-08:00

Zhong et al. (Yale University)* developed an experimental pipeline in C. elegans to identify transcription factor binding sites, using chromatin-immunoprecipitation and deep sequencing (ChIP-Seq). They studied the tissue-specific transcription factor PHA-4 and found distinct sets of PHA-4 targets under conditions of embryonic development and environmental stress (starvation).

The researchers used a GFP-tagged fosmid clone, constructed using the pCC1FOS Vector, to create transgenic worms expressing PHA-4. For starvation assays using RNAi, transcripts were prepared using the AmpliScribe™ T7 High Yield Transcription Kit. The DNA isolated after ChIP was treated with the End-It™ DNA End-Repair Kit before A-tailing, adaptor ligation, and sequencing.

*Zhong M, et al. (2010). Genome-wide identification of binding sites defines distinct functions for Caenorhabditis elegans PHA-4/FOXA in development and environmental response. PLoS genetics, 6 (2) PMID: 20174564

Tangled fragments are “for the birds”: Bird-nesting explained

2010-03-01T05:00:00.000-08:00

Why do the fragment sizes in my Nextera™ library seem longer than the 200-400 bases expected?
We’ve had a few customers ask this question when using the Nextera DNA Sample Prep Kit (Illumina-Compatible). After the Nextera tagmentation reaction and PCR, you may notice an apparent fragment size of 500 bases (or greater) when the DNA is analyzed on an Agilent Bioanalyzer. This phenomenon is called “bird nesting”: the amplified, Transposome™-tagged DNA fragments get intertwined up and appear longer than they really are. This is not a problem. The denaturation step during bridge PCR will eliminate this tangle and the average peak size will settle back down to the expected 200- to 400-base range.

Illumina-compatible sequencing libraries were prepared using Nextera LMW buffer according to the standard protocol. PCR products were purified using either Zymo (red trace) or AMPure beads (blue trace) per the manufacturer’s instructions. The resulting sequencing libraries were examined using a BioAnalyzer (Agilent). The traces show an example of “bird nesting” where noncovalent concatamers result in higher apparent molecular weight under native conditions.

Overview of Nextera™ technology

2010-02-25T06:42:00.000-08:00

Here's a brief introduction to Nextera technology for preparing DNA libraries prior to next-generation sequencing. The presentation also includes recent data from sequencing runs on Roche GS FLX Titanium and Illumina GAII platforms.

For best results, view the slides in full-screen mode.

Nextera Overview Feb 2010

Simplifying library preparation for next-generation sequencing

2010-02-22T13:32:00.000-08:00

According to an industry research report on next-generation sequencing, published in August 2009:

The main bottleneck in sample preparation for next-gen sequencing reported was library construction.
The most used method for DNA fragmentation was nebulization for both short and medium/long fragments, and sonication for fragmentation for ChIP.
The aspect of sample prep for next-gen sequencing most in need of automation was DNA fragmentation.

Nextera™ technology was developed to address the concerns associated with these sample prep methods. DNA sample prep kits compatible with the Roche 454 and Illumina platforms were launched in early January. The kits use in vitro transposition technology to combine fragmentation and tagging of the DNA into a single-tube reaction. The technology will be showcased at the CHI XGen Congress, March 15-19, 2010 in San Diego, CA. A poster on applications of Nextera technology is also being presented this week at the 2010 AGBT meeting in Marco Island, FL.

Creating high-quality metagenomic libraries need not be a dirty job

2010-02-19T14:06:00.000-08:00

The study of metagenomic DNA samples is a hot topic in microbiology, as such samples can shed light on the bioactivity present in many complex environments. Previously, isolation of DNA from complex samples, including soils, compost, and water was not a straightforward process and, in many cases, could result in the loss of genetic information from rare organisms present in a given sample. Recently, Epicentre launched the Meta-G-Nome™ DNA Isolation Kit, which has simplified and streamlined DNA purification from many environmental sample types. The kit generates ~40-kb DNA that can be used directly in construction of a metagenomic DNA library using the CopyControl™ Fosmid Library Production Kit. The DNA is also well suited for PCR screening, or for next-generation sequencing projects, e.g., using Nextera™ sample preparation technology.

Recently, Dr. Marcus Taupp and colleagues in the lab of Dr. Steven Hallam at the University of British Columbia demonstrated the process of making a large-insert metagenomic library, in the Journal of Video Experimentation (JoVE), using the CopyControl Fosmid library Production Kit.

Introduction

2010-02-18T14:00:00.000-08:00

Welcome to EpiCentral, the technical blog by Epicentre Biotechnologies. This blog is maintained by a team of scientists who are dedicated to offering technical support, news, detailed protocols, and novel applications of our products. We hope that you’ll visit us often, add this site to your bookmarks, subscribe to the feeds, and share the information with your colleagues. Suggestions and comments are always welcome!