Friday, June 25, 2010

Functional metagenomics reveals mechanisms of antibiotic resistance

The CopyControl™ Fosmid Library Production Kit has established itself as the molecular tool of choice in studying many facets of environmental metagenomics. Donato et al. at the University of Wisconsin-Madison explored several antibiotic resistance genes from soil microbes in an apple orchard. The study focused on Streptomyces bacteria, long known to be a reservoir of multiple antimicrobial resistance markers. Older techniques, such as cultivation of microbes from soil and determining resistance based on the ability to grow these microbes can fall short in locating these resistance genes, primarily due to the inability to cultivate some of these bacteria. Functional metagenomics, which involves inserting large fragments of foreign DNA into E. coli and assaying the resulting clones for expressed functions, allows the study and isolation of various activities encoded by genes from microbes that are otherwise uncultivatable.

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

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

Tuesday, June 22, 2010

Superior ribosomal RNA (rRNA) removal for RNA-Seq

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

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

Friday, June 18, 2010

Get rid of the small stuff

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

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

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

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

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


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

Tuesday, June 15, 2010

Visit our poster at the CHI Beyond Sequencing meeting

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

Tuesday, June 8, 2010

Library preparation for ChIP-Seq

While Epicentre’s novel Nextera™ technology is revolutionizing next-generation sequencing library preparation, many laboratories are still using older methods of creating genomic DNA libraries for next-generation sequencing. A recent study (Cheung et al.*) of transcriptional regulation mediated by trimethylated histone H3K4 used ChIP-Seq analysis in samples obtained from the human prefrontal cortex.

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

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

Friday, June 4, 2010

From cows to chickadees: BuccalAmp™ Kits in animal genomics

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

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

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

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

Tuesday, June 1, 2010

ASM 2010: A good week for Epicentre

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

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