[GW_OK]

It would appear that I have the most updated protocol. The only place I can see RNase being is in the sureselect elution buffer, which I had assumed was merely a NaOH solution given the immediate use of the neutralization buffer afterwards.

[upenn_ngs]

I suspect the "bait" RNA is digested during the bead wash steps. Also, Agilent furtively releases updates to the sureselect protocol, so make sure you are following most recent, http://www.opengenomics.com/sureselect

[GW_OK]

But a bound bait could still bind DNA though, no? I think it may be a moot point anyway, as my Agilent rep says the baits are destroyed in an RNase step. But I don't see RNase mentioned in the Sureselect protocol I am looking at...

[upenn_ngs]

what exactly do you mean by reuse agilent sureselect? if you are referring to the capture library, then that is a bad idea. the streptavidin beads will pull down all of the biotinylated RNA "baits" whether complementary genomic sequence has been captured or not.

[GW_OK]

Does anyone know if you can reuse agilent sureselect once the baits have been captured by the streptavidin beads?

[upenn_ngs]

Our lab is preparing to set up a similar seq cap pipeline. I'd be grateful to hear any pros/cons or other thoughts you (or anyone else!) have on this system.

simple workflow and covaris shearing is great. pooling for cost efficiency looks to be tricky as the concentration must be very accurate in order to hybridize multiple sample fragments in one reaction.

[GW_OK]

we are using agilent sureselect enrichment to capture sheared dna (200bp, covaris) for resequencing on an illumina platform.

Our lab is preparing to set up a similar seq cap pipeline. I'd be grateful to hear any pros/cons or other thoughts you (or anyone else!) have on this system.

[upenn_ngs]

we are using agilent sureselect enrichment to capture sheared dna (200bp, covaris) for resequencing on an illumina platform. does anyone have experience pooling samples before hybridization with the rna library?

[stanford_genome_tech]

targeted resequencing

Our group at Stanford has developed a targeted genomic circularization approach which is a solution based method. We have applied for resequencing targets up to ~900 kb and have designed an "exome" design set of oligos that cover the CCDS gene annotation.

Cost per sample depends on number of oligos which based on a traditional synthesis lasts >1000 samples. Estimate for targeting assay will be very (<50$) if one amortizes based on several hundred samples.

Hope to release to the biomedical research community soon.

[ATGC_man]

Apparently it's possible to enrich up to ~4.6Mb. You do, however need to buy the equipment - and that's quite expensive. I don't think it's available as a service just now. From what I hear it is going to be an expensive approach to targeted-seq.

Hello,

1. RainDance Technologies supply a system at $225,000 excluding local taxes , insurance and delivery charges

2. RainDance Technologies supply the reagents, chips and consumables at a ‘total cost per sample’ for sequence enrichment as follows;

a. 384 Amplicon library : $600/sample

b. 1,536 Amplicon library : $900/sample

c. 4,000 Amplicon library : $1,200/sample

d. This is the same cost structure for custom libraries or our standard amplicon libraries

3. RainDance Technologies has a Service Provider in the USA who will be receiving their system shortly.

4. They plan to have a European Service Provider engaged in this quarter of 2009

Wait and see...

[brasj]

Apparently it's possible to enrich up to ~4.6Mb. You do, however need to buy the equipment - and that's quite expensive. I don't think it's available as a service just now. From what I hear it is going to be an expensive approach to targeted-seq.

[mrothe]

Capacity of Raindance

Which targeted regions of interest can you amplify with the Raindance technology? Is it possible to enrich 1Mb - 5Mb regions?

[brasj]

Also, Agilent's paper just came out a few days ago. Looks interesting.
http://www.nature.com/nbt/journal/va.../nbt.1523.html

[ATGC_man]

here new Sequence Enrichment Solution from Raindance Technologies...

Someone have hear something about that ?


RainDance Technologies Announces Commercial Release of its Sequence Enrichment Solution
Lexington, MA | Posted on February 5th, 2009
In an announcement at the 2009 Advances in Genome Biology and Technology Conference (AGBT), the company said it has begun accepting orders for the solution and has started commercial production of the solution's components for shipment during the first quarter.

"This milestone represents the first commercialized application of our exciting RainStormTM microdroplet-based technology platform," said Chris McNary, President and Chief Executive Officer of RainDance Technologies. "It underscores the power of droplet-based biology to increase the depth of understanding about biological problems and increase the pace of biomedical discovery."

The RainDance Sequence Enrichment Solution incorporates RainStorm technology for targeted sequencing applications of the human genome. It will enable the high-resolution analysis of genetic variation between individuals within populations at a level unmatched by current methodologies. The solution dramatically improves sample uniformity and reduces the selection bias typically associated with targeted sequencing.

The Sequence Enrichment Solution consists of the following components:

* The RDT 1000 system generates picoliter-volume discrete PCR reactions in droplets at the unprecedented rate of 10 million per hour. It amplifies hundreds to thousands of genomic loci with high specificity and uniformity.
* Custom-Order Content for DNA Primer Libraries will be available that enable the amplification of hundreds to thousands of genomic loci in a single tube. RainDance customers can design and order these libraries specifically for their research applications.
* Sequence enrichment consumables kits, provide a complete workflow solution for easy implementation in the laboratory.

The company also announced that comprehensive training and support services are available to maximize the operating performance of the solution.

"This commercialization step is the culmination of a rigorous Early Access Partner program that involved some of the world's leading genomic research institutions," said McNary. "These organizations see the potential of our Sequence Enrichment Solution to enhance the performance of their next-generation sequencing platforms and accelerate their discovery process."

RainDance also is presenting a workshop, "Enrichment of Genomic Loci for Targeted Sequencing Applications," at the AGBT Conference on February 6 to explain the application and benefits of its new technology in genomics research.

For sales information, contact [email protected] or call 781-861-6300. The company said the Sequence Enrichment Solution will be available for purchase in Europe later this year.

####

About RainDance Technologies, Inc.
RainDance Technologies Inc. is a provider of innovative microdroplet-based solutions for human health and disease research. The speed and simplicity of the company’s exciting new technology platform enable researchers to design experiments in ways that were previously unaffordable or unimaginable. The company’s RainStormtm technology produces picoliter-volume droplets at a rate of 10 million per hour. Each droplet is the functional equivalent of an individual test tube and can contain a single molecule, reaction, or cell. This versatile technology can adapt proven assays for high-speed workflows with minimized process-induced bias or error.

RainDance’s initial application will focus on the targeted sequencing of the human genome — one of the fastest-growing segments of the $1 billion DNA sequencing market. This application will enable the high-resolution analysis of genetic variation between individuals and populations at a level unmatched by current methodology.

RainDance was founded in 2004 by scientists from Harvard University; the Medical Research Centre in Cambridge, England and the ESPCI in Paris.

RainStormtm is a trademark of RainDance Technologies, Inc.

[brasj]

I'm just thinking of GC rich regions (TM differences); amount of captured sequences per run (I think it will be difficult to get to the 30Mb advertised by nimblegen); polymerase errors...

I may be completely wrong, but my feeling is that PCR just isn't it... at least for large projects; may be great for smaller (low number of capture sequences) projects.

what do you guys think?