For NRC Team, Pippin Platform Leads to Higher-Quality Assemblies
At the DNA Technologies Laboratory at the National Research Council of Canada, scientists are using the Pippin size selection platform to improve the quality of their genome assemblies.
Andrew Sharpe, Research Officer and Group Leader of the Saskatoon-based laboratory, got his first Pippin Prep last year. He added a second Pippin Prep as well as the longer-fragment Blue Pippin to his arsenal earlier this year.
In Sharpe’s lab, which also serves as a core facility for NRC and other Canadian government agencies, assembly projects tend to focus on large plant and fungal genomes. His team relies on Illumina and 454 sequencing, often adopting a hybrid assembly approach to take advantage of both platforms.
“The majority of libraries going through are the shorter, standard pair-end libraries of 200 to 400 bases,” Sharpe says, noting that those libraries run on all three Pippin machines. Longer mate libraries — usually in the range of 3kb to 10 kb — are also a good fit for the Pippin, he adds.
Sharpe and his colleagues use the Pippin platform to create multiple pair-end libraries for the same sample — constructing, for instance, three libraries with 200-base, 300-base, and 400-base inserts — and then assemble all of those sequences together, often using SOAPdenovo. “If you assemble one of the libraries, then you’ll end up with an assembly. But if you assemble all three together using three different lengths, you get quite a bit better product,” Sharpe says. “The nice thing with the Pippin Prep is being able to easily get those discrete size ranges.”
Before Sharpe had the Pippin, his team spent a lot of time on manual gel extractions. “Having the Pippin makes things quite a lot more efficient on the labor side,” he says. Now he’s looking to the Blue Pippin to take over for the field inversion gel electrophoresis his team runs for making larger 454 mate libraries. “The Blue Pippin offers the prospect of actually speeding up that process and hopefully getting away with less amounts of DNA,” Sharpe says. “You normally need a lot of DNA to operate on the standard FIGE gel, but with the Blue Pippin we should be able to get away with less.”
BluePippin:Collecting all DNA “Greater Than” 1kb
We have received several requests to use the Pippin to collect all remaining DNA above a programmed base pair value from a sample. Although Pippins currently have the capability to collect all DNA after a run time threshold (using the “Time” programming mode), there is no method to elute the entire sample after a programmed base pair value . Also, our protocol editor requires users to enter an ending base pair value (BP End) in the “Range” mode and will not accept values above 50,000 bp.
For the BluePippin, we have developed a protocol for this purpose, and named the cassette file “0.75% Greater Than – Marker S1”. This requires our 0.75% dye-free gel cassettes kit for lower ranges (BLF7510). With this protocol, users enter a 4 hour run time, and enter a 50,000 bp value into the “BP End” field using the “Range” programming mode. The 50,000 is a dummy value that tells the instrument to continue collecting until the end of the run.
At this time, the “0.75% Greater Than – Marker S1” cassette file is not available in the standard menu of cassette types, but we can provide it to you separately. Contact us if you are interested.
An example of “Greater Than” collections:
Broad Institute Teams with Sage Science for Automated Sizing
Don’t miss this great blog post from the Broad Institute (“A Sage partnership”) describing collaborative work between their genome sequencing team and Sage Science to design a better size selection process for the Broad’s sequencing pipeline.
Headed up by Sheila Fisher, assistant director of technology development for the Broad’s Genome Sequencing Platform, the goal was to replace error-prone, tedious manual gel extractions in the sample prep workflow. Working with Sage’s Pippin platform, Sheila and her team were able to automate the size selection step, improving accuracy and eliminating the chance for cross-sample contamination.
An added bonus was that Pippin sizing offered much higher yields than manual gel extraction had, allowing Fisher’s team to accept samples with just 100 nanograms of DNA, instead of the 3 or 4 micrograms the pipeline originally required. “This opened up a significant number of samples to the process that we couldn’t sequence before,” says Sheila in the blog post. “We were able to build a very strong partnership with Sage, and the result was a true co-development project.”
We couldn’t have said it better ourselves. It’s truly a pleasure to continue with our great collaboration with the Broadies!
Automated Size Selection Key for RADseq: PLoS One Publication
Congratulations to Sage Science customer Hopi Hoekstra and her team at Harvard University for their recent publication in PLoS One! Dr. Hoekstra, who works in the Organismic & Evolutionary Biology and the Molecular & Cellular Biology departments at Harvard, reports a full laboratory protocol for RADseq, or reduced-representation genome sequencing for use in population genotyping. The method allows for studying hundreds of thousands of markers across hundreds of individuals or more.
The paper, published on May 31 and entitled “Double Digest RADseq: An Inexpensive Method for De NovoSNP Discovery and Genotyping in Model and Non-Model Species,” can be found here. (link to paper)
The authors write: “Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours.”
As part of the library prep protocol, the Harvard team tested out manual gel extraction versus the Pippin Prep for size selection. The paper reports that manual gel excision did not perform as well as automated size selection, “likely because [gel excision] was imprecise or ‘leaky.’” The authors note that for manual gel electrophoresis, “careful practitioners can achieve roughly 50% of the precision and repeatability of automated DNA size selection.”
For more on the Hoekstra lab, click here. (link to her lab)
At Emory, Size Selection Saves Time for NGS Prep
At the genome center at Emory University, scientists credit the Pippin Prep with shaving almost a full day off the sample prep process for Illumina’s mate-pair library prep.
Chad Haase, laboratory manager at the Emory GRA Genome Center, says that the Sage Science Pippin Prep size selection instrument his lab acquired about a year ago has replaced the 16-hour runs that had previously been done on gels with a very low agarose concentration.
Jamie Davis, a scientist at the genome center, says that she typically runs the Pippin “after we do our first end repair and the biotinylation reaction.” Size selection generally takes about an hour, she says.
The Pippin Prep also works well for 454 sequencing. Haase’s team had tried another automated gel system, but it wouldn’t work for fragments larger than 1 kb. “Once we started making the large mate-pair libraries — 3 kb, 8 kb, and 20 kb fragments — then we had to come up with something different and the Pippin system was the best,” he says.
“With the large kb mate-pair libraries, the Pippin has really been our savior there,” Haase says. “A sample prep process that used to take us three days, we can now get down to roughly two days.”
Haase says that his team also uses the Pippin before amplicon sequencing on the Roche/454 GS-FLX Titanium. “If you have any small fragments whatsoever in a 454 amplicon library, it’s going to ruin the run,” he says. “The emulsion PCR prior to sequencing is preferential to smaller fragment amplification, so you’d get a whole bunch of 50-base-pair sequencing reads” without using the Pippin for size selection.