Thursday, December 22, 2011

Large-insert cloning aids study of dinoflagellate species

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.
ResearchBlogging.orgJaeckisch, 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

Monday, December 12, 2011

What's new in the ScriptSeq v2 kit?

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.

Friday, December 9, 2011

Role of Piwi proteins in mammalian transposon silencing

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.

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

Friday, December 2, 2011

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

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.

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

Friday, November 18, 2011

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

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:
  1. RiboMinus treatment may require two rounds of rRNA depletion whereas Ribo-Zero treatment only requires round of depletion.
  2. 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%.)
  3. 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.
  4. 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.
ResearchBlogging.orgHuang, 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

Wednesday, November 9, 2011

Medicinal leeches, MasterPure RNA Kits, and metatranscriptomics

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.

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

Friday, October 21, 2011

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

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

Tuesday, October 11, 2011

Illumina launches Nextera DNA Sample Prep Kit

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.

Tuesday, September 27, 2011

Preparing high-quality DNA and RNA for Illumina sequencing

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 (A260/280 >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

Wednesday, September 14, 2011

Nextera libraries aid in study of honey bee pathogens

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.

ResearchBlogging.orgRunckel, 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

Friday, September 2, 2011

Nextera library prep used to characterize novel virus variants

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.

ResearchBlogging.orgLauck, 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

Thursday, August 18, 2011

Nextera technology enables complete sequencing of the CHO cell genome

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.

ResearchBlogging.orgXu, 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

Thursday, August 4, 2011

Fosmid libraries aid in identifying a novel lipase

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.

ResearchBlogging.orgGlogauer, 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

Wednesday, July 27, 2011

Species compatibility for Ribo-Zero rRNA Removal Kits

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.

Friday, July 8, 2011

EZ-Tn5 Custom Transposon Contruction Kits now available

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.

Wednesday, June 29, 2011

Something old, something new: Nextera and CopyControl technologies

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.

ResearchBlogging.org1. 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

Thursday, June 23, 2011

Summer stock-up special

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

Monday, June 6, 2011

Nextera technology featured at ASM 111th General Meeting

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.

Friday, May 13, 2011

Visit Epicentre at the ASM 2011 General Meeting

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!

Tuesday, May 10, 2011

Ribo-Zero rRNA removal from FFPE RNA

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.

Friday, April 1, 2011

Nextera library primers demystified

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.

Wednesday, March 16, 2011

Preparing Nextera libraries from short linear DNA

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.

Wednesday, March 9, 2011

Visit Epicentre at the CHI XGen Congress

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.

Thursday, February 10, 2011

Circligase II-based detection of miRNAs

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.

ResearchBlogging.orgKumar, 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

Tuesday, January 18, 2011

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

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."

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

Wednesday, January 12, 2011

Illumina acquires Epicentre Biotechnologies

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

Monday, January 10, 2011

Visit Epicentre at the PAG XIX Conference

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
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.

Thursday, January 6, 2011

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

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.

ResearchBlogging.orgLamm, 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