“We have found that the BluePippin™ size selection platform is very useful for applications where long read lengths are important, such as de novo genome assembly,” said Kevin Corcoran, Senior Vice President of Market Development at Pacific Biosciences. “Our studies have demonstrated that size selection with Sage’s BluePippin platform is an easy addition to our customers’ sample prep workflow that selects for 10 kb or larger fragments for sequencing on the PacBio RS II.”

Based on PacBio’s revolutionary SMRTSequencing technology, the PacBio RS II can generate average read lengths of 5,000 base pairs, with the longest reads above 20,000 base pairs in length. The PacBio RS II System, including consumables and software, provides a simple, fast, end-to-end workflow for SMRT Sequencing in research areas including infectious disease and microbiology, agriculture, and complex genetic diseases with repeat expansions.

The BluePippin platform, part of the Pippin line of automated size-selection tools offered by Sage Science, uses pulsed-field power to take fractions of DNA from fragments ranging from 50 bases to 50 kilobases. Independent studies of size-selection methods for next-generation sequencing sample prep have repeatedly found that the Pippin platform offers unparalleled reproducibility, accuracy, and sample recovery. By eliminating low-molecular-weight templates from sequencing libraries, automated size selection allows users to load longer fragments and boost the average read length produced by sequencing.

“This is excellent validation of the tremendous utility BluePippin offers for libraries designed to generate extraordinarily long sequence reads,” said Alex Vira, Director of Marketing for Sage Science. “We look forward to working with PacBio and its customers as they continue to deliver industry-leading read lengths and conduct countless studies that would be impossible with shorter-read technologies.”

In data presented at the Advances in Genome Biology & Technology conference earlier this year, Pacific Biosciences scientists assessed three libraries (bacterial, fish and mammalian) generated with and without BluePippin size selection. They reported that N50 subread lengths increased by 90%, 110%, and 86%, respectively, in the libraries that were size selected with BluePippin compared to those that were not.

About Pacific Biosciences
Pacific Biosciences of California, Inc. (Nasdaq:PACB) offers the PacBio^® RS II DNA Sequencing System to help scientists solve genetically complex problems. Based on its novel Single Molecule, Real-Time (SMRT^®) technology, the company’s products enable: targeted sequencing to more comprehensively characterize genetic variations; de novo genome assembly to more fully identify, annotate and decipher genomic structures; and DNA base modification identification to help characterize epigenetic regulation and DNA damage. By providing access to information that was previously inaccessible, Pacific Biosciences enables scientists to increase their understanding of biological systems. More information is available at www.pacb.com.

The new SMRTbell^TM 20kb sample prep protocol using the BluePippin is posted on the PacBio^® SampleNet site, click here to view the page or download the document. Instructions for using the BluePippin High Pass cassette definition for the SMRTbell protocol can be found here.

Original Post:
Many thanks to all the scientists who stopped by the Sage suite during the Advances in Genome Biology & Technology meeting in Marco Island — we were thrilled to see you! When we weren’t busy handing out glowing medallions, the Sage Science team had a great time catching up with people, attending the excellent scientific presentations, and walking the halls of the poster sessions.

Along with other attendees, we were really impressed by the posters and presentations showing data from the PacBio^® RS. A number of talks referred to the company’s single-molecule, real-time (SMRT^®) sequencing, which generates extremely long reads — one scientist said he’d produced a 26 kb read on the PacBio RS. Between the read length and unique base modification detection capability, the sequencing platform is particularly useful for de novo assembly, genome finishing, SNP discovery, and epigenetic analysis.

In its quest to produce sequence from even longer DNA fragments, PacBio recently tested our BluePippin platform for size selection; the results of those studies were the basis for a poster we displayed at AGBT. In it, the PacBio authors demonstrate a doubling of the N50 subread length – the value at which 50% of unique DNA bases are in reads longer than this value – when using the BluePippin platform compared to the standard workflow (click to enlarge this excerpt from the poster):

For generating 20 kb libraries that are optimal for the platform’s current polymerase, the authors note that “when combined with size selection to remove the shorter fragments that tend to load more favorably than long fragments, these libraries show greatly increased subread lengths.”

Looking at three examples of libraries for bacterial, fish, and mammalian DNA, the authors report that the N50 subread lengths increased by 90%, 110%, and 86%, respectively. Some of this data was also included in an AGBT plenary presentation by Jonas Korlach, Chief Scientific Officer of PacBio, who cited the BluePippin platform as one of the new improvements for PacBio customers.

Some PacBio customers are already using the BluePippin platform, and we look forward to working with more of them in the coming months. Together, these technologies offer a great and unique solution for truly long-read sequencing.

We’ve set up this post to provide Pippin Pulse users with a resource to keep updated on new protocols and to post references that might be useful. We also invite users to feel use this page as a forum, if you see fit, and we’d enjoy showing interesting gel images and data (email these to support@sagescience.com).

Our go-to book on theory:
“Electrophoresis of Large DNA Molecules:Theory and Applications” (E. Lai and B. Birren, eds), Current Communication in Cell & Molecular Biology Vol. 1,. Cold Spring Harbor Laboratory Press, New York, 1990

Here’s nice overview on pulsed field techniques:
Pulsed Field Electrophoresis for Separation of Large DNA

Download the Pippin Pulse user manual here

Our Gel Set-up
We use a Galileo Model 1214 RapidCast^TM Mini Gel Unit running 12 X 14 cm gels using Lonza SeaKem^® Gold Agarose. We use our Pippin Tris-TAPS buffers (which you can order directly from us in the US and Canada, Part No. KBB1001, under “Accessories” on our ordering page) or 0.5X TBE. The formulations can be found in the Pippin Pulse user manual or separately here.

Gel images of our 1-50kb and 3-70kb protocols:
(click to enlarge images)

What is it?
This definition improves the sample recovery for customers using BLF7510 Marker S1 cassette kits for DNA size selection, for size ranges between 3- 10 kb. The original cassette definition for this range, “0.75% DF 3 – 10kb Marker S1” uses pulsed-field during elution (size selection), and we have subsequently found that direct-current elution improves recovery.

Who should use it?
This is now the recommended cassette definition to use for size-selecting between 3-10kb. However, if you have optimized a protocol using the current version you may want continue to use it (it will not be deleted as a cassette definition option) if you do not wish to re-optimize.

Also note that since the BluePippin system does not allow for simultaneous pulsed-field and direct-current operation, lanes that are being separated using pulsed-field will temporarily be halted during every elution process. This makes the total run times lengths dependent on the elution times (and size selection targets), and therefore more difficult to estimate and can add up to 40 minutes to a cassette run.

How much improved is the recovery?
Our validation of the change in the cassette definition shows up to a 2-fold increase in recovery. As you may know, for collections of large fragments (on 0.75% agarose cassettes) we provide Tween solution which we find improves recovery if one employs a post-collection Tween rinse. Considered together, here are some results from our study. We started with a 5 ug of restriction digested DNA, and 8kb “Tight” collections:

Clark is the sequencing technology development leader at TGAC, where part of his role involves testing out new platforms, deciding whether they’d be a good fit at the institute, and ironing out best-practice workflows for the ones that are chosen.

The BluePippin automated size selection platform from Sage Science is one of the tools that succeeded in Clark’s technology proving ground and is now used more broadly throughout TGAC, where major projects include sequencing the wheat genome and studying honeybees. Clark, who joined the institute in 2010 after spending seven years as a scientist at the Wellcome Trust Sanger Institute, says that TGAC researchers rely on Pippin for building long-insert libraries for sequencing projects.

These projects include de novo sequencing, for which precise size selection in mate-pair libraries offers a significant improvement in quality of the genome assembly. The mate-pair sequence might represent a small fraction of the total data generated for the assembly — most will come from the shorter-insert paired-end libraries — “but it has a massive effect on the quality of the output,” Clark says. “The bigger-insert library gives you a 5x or 10x jump in quality, maybe even bigger, in terms of the sizes of the assembly that you’re able to generate.” These libraries, which are sequenced on Illumina or Pacific Biosciences instruments, offer longer-range information that can fill gaps or jump over repeats and other problematic structures that would otherwise break up an assembly. Indeed, Clark says the TGAC bioinformatics team prefers Pippin-aided sequencing libraries because the tight size selection helps them determine how far apart certain reads should be and put together a more accurate assembly.

Clark’s team had tested other automated size selection options, but Pippin was the best platform for generating these valuable large-insert libraries. (Pippin Prep can yield libraries with inserts up to 8 kb, while BluePippin works for even longer inserts.) It also allows for loading more material than other size selection alternatives, making it a good fit for the no-PCR paired-end libraries that some TGAC scientists like to run, Clark says. “If you have enough DNA, you can just skip the PCR — and you get much better coverage across the genome and a better assembly. That’s easier to do on a Pippin.”

The scientists tried a number of tools — including qPCR, FISH, and Southern blots — but none of the assays could quickly and reliably report the number of gene copies in a mouse. So the team resorted to what DuBose calls “an invention of necessity” and made a custom microarray to identify the site of transgene integration and develop a genotyping assay that could deliver the results they needed.

The results of that work were published in Nucleic Acids Research in a paper entitled “Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene.” Lead author DuBose and her colleagues analyzed regions of the transgenic mouse genome next to BAC sequence by using arrays to capture the DNA, size selecting with the Pippin Prep, and sequencing on the Illumina HiSeq.

DuBose, Collins, and the other authors found that the BAC had broken more than expected when it was injected into the mouse; there were four mouse-human junctions. “I was surprised by how rearranged it was,” DuBose says, noting that the junctions within the BAC were quite unexpected. “We know it’s not unique to our model. I have a feeling that all of the transgenic models have crazy rearrangements — it’s just that nobody goes in to look this closely.”

Ultimately, DuBose used the sequence data to design a PCR assay with three primers crossing one of the BAC-mouse junctions. That assay is now a standard tool that can be used to determine gene copy number in the model mice: results indicate whether a mouse is homozygous mutant, heterozygous mutant, or wild type.

The lab has now returned to its HGPS research, but DuBose says that the technological approach to characterizing a transgene and developing a PCR genotyping assay can be used by anyone working on transgenic models or genome sequence that has foreign DNA in it. She notes that the array her team used for targeted capture is no longer available, so the technique would have to be adapted for liquid capture.

MiRNAs are especially tricky to extract from gels because the band of interest is near other bands containing unwanted products that adversely affect the quality of the sequencing run. “When you look at a Bioanalyzer trace, the band we’re after really is pretty small,” says Jennifer Bair, a member of Knudtson’s lab. “You see a lot of the other peaks much better.” The miRNA range of interest might be just 145 bases to 160 bases in size, but there will be quite a lot of adapter-dimers, primers, and other smaller content, as well as RNAs larger than the desired target. “We’re always amazed that the Pippin can isolate such a small fraction with such accuracy,” Bair adds. “It’s great.”

“For a microRNA process, there are a couple of bands that you do not want to collect,” Knudtson says. “Effectively excluding the unwanted or contaminating bands is essential to having good-quality data.”

The core facility brought in the Pippin Prep size selection instrument from Sage Science last year to replace manual gels, which are time-consuming and prone to inconsistency. “Cutting a slice out of a gel is a very subjective thing to do, and it can be technically challenging to repeat that same cut,” Knudtson says. “But when we run these out on the Pippin Prep, we’re getting exactly what we hope to receive. I can have different technicians do the same procedure and essentially get the same band or answer back when they’re processing samples.”

Knudtson’s team deploys automated sizing for more than the miRNA workflow. It has been useful for next-gen sequencing sample prep in the Iowa lab, which has a range of instruments: 454, HiSeq, MiSeq, and PGM. It takes a lot of samples to feed that pipeline, Knudtson notes. “Anything we can do to streamline the workflow is appreciated,” he says. “The Pippin Prep helps to shorten that process — it has sped up the turnaround time of samples.” Automated size selection also contributes to more efficient loading of flow cells, letting Knudtson get the most bang for his buck with sequencing runs.

The scientists have also begun to use the Pippin for ChIP-seq experiments, in which shearing chromatin to the correct size can be a real challenge, says Bair. While they are still evaluating how much size selection helps with this particular process, Bair says that generally the libraries can be made more efficiently without the larger fragments that sometimes get incorporated during the ChIP pulldown step.

Knudtson is pleased to have the days of manual gels behind him. From now on, he says, “if the protocol requires a gel extraction, we’re going to use the Pippin Prep to do it.”

“Evaluation and optimisation of preparative semi-automated electrophoresis systems for Illumina library preparation” from lead author Michael Quail and colleagues at the Wellcome Trust Sanger Institute, demonstrates several types of comparisons between the systems. The authors note in their premise that better sizing techniques are needed since manual gels are time-intensive and lack reproducibility while SPRI beads often give ranges “too broad for de novo assembly applications.” The authors assessed size selection methods for general library preparation, PCR bias, noPCR use, and more.

The importance of accurate size selection goes beyond removal of small fragments, including adaptors and adaptor-dimers, to improve de novo sequence assembly and allow for chromosomal rearrangement prediction, the authors note.

In their first test for whether the instruments performed according to their specifications, “both platforms collected the specified size fractions but with quite different results,” Quail et al. write. “The Pippin Prep gave very tight size distributions whereas those from the Labchip XT were much broader. … The distribution of recovered fragment sizes was much more reproducible with the Pippin Prep.” Such tight sizing can lead to lower yield, the authors note, adding that “this can partially be overcome by setting the instrument to collect a wider size range.”

The study also looked at the effects of performing size selection prior to PCR amplification, finding that the tightest possible size fraction was advantageous before amplification. “Whilst Pippin Prep eluted DNA, size selected using the ‘tight’ setting, gave sharp and representative size distributions following PCR, we regularly observe smaller fragment ‘shoulders’ … following amplification of Labchip XT fractionated ligation products,” the scientists write.

While this paper focused on sample prep for the Illumina sequencing workflow, the authors note that Pippin works well with other next-gen technologies too. “We have found the Pippin Prep to be useful for both size fractionation of 100bp and 200bp fragments for the Ion Torrent PGM, and for size fractionation of 3kb fragments for Pacific Bioscience sequencing,” they write.

ScriptSeq Complete kits are designed for the highest quality stranded RNA-Seq sample prep; they allow scientists to remove ribosomal RNA from total RNA samples and build libraries with >98% directionality.

The new cassettes for Pippin Prep have smaller elution wells to support the volumes (25 µl) used in the ScriptSeq Complete protocol. Libraries purified by Pippin Prep show ~10-15% increase in library diversity compared to the bead-based method. (Library diversity was measured in number of genes, transcription start sites, isoforms, splice sites, and promoters identified in sequencing results.)

Epicentre has included Pippin as the most recent purification option for the ScriptSeq Complete kits. For the full protocol, click here or visit www.epicentre.com/pippin for more information. Pippin Prep users should use part number SSQ2010 to order gel cassettes.

Levine joined the BioMicro Center about four years ago; in that time, he has transformed it from a two-person lab to a major genomics and bioinformatics core facility with a dozen full-time employees serving more than 80 MIT faculty members. “My goal was to create an integrated core which would work with labs on everything from experimental design through to experimental analysis and anything they need help on anywhere along that chain,” Levine says.

His team of technology experts supports faculty members from a number of departments and institutes, covering scientific areas as diverse as cancer, environmental health sciences, biological engineering, and more. Levine’s core lab colleagues are professional scientists who focus on tools and methods, so they can translate that expertise across scientific areas and tailor experiments to each customer. “Ultimately what comes into our lab is nucleic acid, and what we do with nucleic acid is relatively constant,” Levine says. “The technology improvements that are useful for, say, understanding the evolution of ocean ecosystems can also apply to cancer research.”

In the last few years, Levine added sequencing sample preparation steps, and says that he realized early on he would have to find alternatives to manual gels. As a chargeback facility, Levine has to base prices on the fully-loaded costs of any service. When it came to setting prices for manual gel procedures, “I had to budget how long it takes to pour a gel, run out the sample — one per gel to avoid contamination — cut out the band, isolate the band. And when I put a price tag on it, I know that price tag is high enough that absolutely no one will pay for it. Once you start adding in the labor costs, the economics don’t make any sense at all,” he says.

Levine has been using the SPRIworks System from Beckman Coulter Genomics, which has been very successful as a gel alternative. However, certain applications require tighter sizing than was possible with that system, which is why he decided to look at the Pippin platform.

“What Pippin let us do was get into areas that we hadn’t been able to before,” Levine says. Two of those areas were RNA-seq — particularly identifying splice variants — and miRNAs, he adds. On the miRNA front, his team would otherwise have had to use manual gels and cut out bands, but “we wouldn’t be able to do that with any kind of economy of scale,” he says. “The high percentage gels on the Pippin allow us to cut out bands of the right size for microRNAs.”

When it comes to splice variant analysis, Levine says that his team recommends very tight sizing. “Some of the RNA-seq methodologies, when you’re doing de novo sequencing of transcriptomes and want to do assemblies, tend to perform better when the size distribution of the library inserts is very tight,” he says.

The reason this is helpful for splice detection is in adding another dimension of data in the alignment step to allow for the inference of structure between known nucleotides. “If you know where the left read is, and where the right read is, and if you know the size of the fragments, then you can infer based on known exons the entire pattern in between,” Levine says. “You can calculate the likelihood that the exon is included or not based on where pairs of reads are.” The same holds for de novo assembly in general, he notes. With very tight size distribution, “you have a much more constrained situation when you’re assembling,” he adds.

Levine also believes that the Pippin platform is well suited for planning ahead as sequencing technologies evolve to offer longer reads. “As we need longer inserts, we’re going to be limited in terms of what we can get off the SPRIworks machine,” he says. Pippin’s ability to work with longer ranges means that “it’s extremely useful both for the ability to do sample preparation now and for the ability to work with these future technologies.”