Tuesday, March 30, 2010

Nextera™ tagmentation of soil metagenomic DNA

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

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

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

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

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

Tuesday, March 23, 2010

Nextera™ libraries and sequencing primers

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'

Thursday, March 18, 2010

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

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

Monday, March 15, 2010

Lower prices, larger sizes for Nextera™ kits

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).

Wednesday, March 10, 2010

Nextera™ technology at the CHI XGen Congress

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.

Monday, March 8, 2010

Extracting DNA or RNA from FFPE Tissues

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

Thursday, March 4, 2010

ChIP-Seq used to examine diverse roles of transcription factors

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

Monday, March 1, 2010

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

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.