Sage Blog

Illumina Workflow: Pippin for RNA Studies

Illumina sequencers are far and away the most popular platform on the market. Since so many Sage customers use their Pippins with these instruments, we’re taking a look at some of the most common or interesting applications of automated DNA sizing with the HiSeq, MiSeq, and even the GA II or GA IIx workhorses. If you missed it, check out our recent blog about using Pippin for Illumina mate-pair sequencing. Our focus today is RNA studies, from RNA sequencing to microRNAs and other small RNAs.

If you’re using the MiSeq or HiSeq for RNA studies, chances are good you’re deploying a kit such as ScriptSeq from Epicentre. We offer a cassette custom-tailored to the ScriptSeq Complete™ kits with smaller elution wells that match the volume (25 µl) needed for the rest of this workflow. Sizing is essential to this protocol because it increases library diversity. A study of Pippin Prep with these kits showed a 10 to 15 percent boost in library diversity (measured in number of genes, transcription start sites, isoforms, splice sites, and promoters identified in sequencing results) compared to bead-based sizing.

Epicentre includes Pippin in its protocol for the ScriptSeq Complete kits. Check out the documentation here or visit www.epicentre.com/pippin for more information. Pippin Prep users should use part number to order gel cassettes.

Pippin also proves useful in small RNA studies, such as miRNAs. Customers like Kevin Knudtson from the University of Iowa and Stuart Levine at MIT say that Pippin Prep is an integral part of the miRNA pipeline. Knudtson, who runs the university’s DNA Facility, says his team will not even “consider a manual gel extraction for microRNAs.” In their hands, Pippin is remarkably effective at isolating miRNAs, even with a lot of adapter-dimers, primers, and other small content in the neighborhood.

According to Levine, director of MIT’s BioMicro Center, “The high percentage gels on the Pippin allow us to cut out bands of the right size for microRNAs.” In a core facility, the alternative — manually cutting bands from a gel — is not economically feasible. His team also uses Pippin Prep for splice variant analysis with an RNA-seq workflow. “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.

We also worked with New England Biolabs as they developed their NEBNext Multiplex Small RNA Kit, which functions best with Pippin sizing. A recent paper from scientists at the Mayo Clinic, Institute for Systems Biology, and University of Illinois demonstrated this workflow in a project examining the effects of vitamin D on microRNA regulation of gene expression in zebrafish. They enriched for miRNAs of interest with Pippin and the NEBNext kit. To learn more about how these products fit together, check out this app note.

Our blog series will continue with peeks into how scientists are using Pippin and Illumina sequencers together for ChIP-seq, ddRADseq, and more. Check back soon for more!

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Poster on Full-Length Transcript Sequencing Includes SageELF

We are pleased to report the first customer poster featuring the newest addition to our product lineup, the SageELF whole-sample fractionation tool. “Single Molecule, Real-Time Sequencing of Full-length cDNA Transcripts Uncovers Novel Alternatively Spliced Isoforms” comes from scientists at Pacific Biosciences and the University of Washington.

The poster describes a human gene expression study in which the scientists were able to generate full-length isoform sequences, with some transcripts longer than 10 Kb. Isoform sequencing was conducted using the PacBio® RS II DNA Sequencing System. The cDNA sequences came from the MCF-7 human breast cancer cell line as well as from brain, heart, and liver cells.

The authors note: “Even in extensively profiled sample types, the method has been able to uncover large numbers of novel alternatively spliced isoforms and previously unannotated genes.” This ability to produce full-length transcript sequences offers a unique way to examine alternative splicing in eukaryotic organisms.

The team used SageELF, an automated sample prep device that generates 12 contiguous fractions from a single DNA or protein sample. It can be used in NGS workflows to build libraries with multiple insert sizes from the same sample, as well as to preserve precious samples. This Bioanalyzer trace of SageELF fractions is excerpted from the scientific poster:

bioanalyzer ELF from Clark poster

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Illumina Workflow: Pippin or SageELF for Mate-Pair Sequencing

Most Sage customers use their Pippins with an Illumina workflow, so for the next few weeks we’ll be taking a look at some of the most common or interesting applications of automated DNA sizing with the HiSeq, MiSeq, and even the GA II or GA IIx workhorses.

Today we look at mate-pair sequencing, a protocol using a large insert between reads to cover greater genomic distances. The approach is used to span highly repetitive regions and can result in longer contigs and fewer assembly gaps. Often, mate-pair sequence data is combined with shorter-insert paired-end reads to cover a genome more thoroughly. It is used for de novo sequencing as well as structural variant detection and genome finishing.

For Illumina sequencing, the Pippin Prep and BluePippin save time and provide a reproducible alternative to manual size selection that will ensure better sequencing results. Mate-pair sequencing can be tricky, so the more precise your library sizing, the more accurate your data will be in the end. If you’re performing the newer mate-pair sequencing with Nextera, Illumina recommends using the Pippin platform to get “more stringent” sizing than can be accomplished with AMPure alone. (We’re under Size Selection in Chapter 3, beginning on page 40 of the guide.) The document reports that “in our experience running a standard agarose gel does not provide as robust and reproducible results as the Sage Pippin Prep.” We’re honored to be in the official guidelines!

A paper from scientists at the Wellcome Trust Sanger Institute last year offers another take on the mate-pair protocol. The publication, “An improved approach to mate-paired library preparation for Illumina sequencing,” describes optimized techniques to boost library complexity and quality while reducing the occurrence of chimeras. The technique was designed to improve mate-pair success when not much DNA is available or the sample is degraded. BluePippin is a critical part of the final workflow. While the authors noted that other size selection methods could be used, they note, “The Blue Pippin provides the greatest recovery and accuracy of currently available commercial methods.”

Mate-pair sequencing is a great fit for our newest instrument, the SageELF, which performs whole-sample fractionation. SageELF generates 12 contiguous fractions from a DNA sample, allowing scientists to build short-insert and long-insert libraries from the same sample.

Our blog series will continue with peeks into how scientists are using Pippin and Illumina sequencers together for microRNA studies, ChIP-seq, ddRADseq, and more. Check back soon for more!

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ASMS in Review: Proteomics and Posters Galore

The Sage Science team headed back to our home base of Beverly, Mass., after four terrific days at the ASMS conference in Baltimore. The scientific content of the show was so good that we didn’t even mind enduring the heat wave that engulfed the city.

We spent a great deal of time absorbing the hundreds of posters presented during the meeting. There were some really impressive scientific efforts, ranging from the very large — such as cataloging the human proteome — to the very small, including nanomaterials.

A poster that caught our interest came from scientists at the Technical University of Munich and other organizations. It described Proteomics DB, a public database containing more than 90 percent of human proteins. (For more in-depth info, check out this article from The Scientist.) This draft of the human proteome, including novel proteins from supposedly noncoding portions of the genome, was generated by compiling existing mass spec libraries along with newly created libraries based on dozens of tissue types, sera, and cell lines. We are real fans of the concept of creating these comprehensive databases, and are proud that building a similar resource for E. coli was the first external use of our SageELF tool.

Another poster highlighted work from the University of Texas and MD Anderson, which demonstrated the use of mass spec together with RNA-seq to get a multidimensional view of cancer stem cells. And in separate work similar to the SDS removal tool we saw earlier in the week, scientists from Dalhousie University in Nova Scotia are developing an interesting technology using plastic columns to precipitate proteins on a Teflon disc.

Thanks again to all of the attendees who stopped by our booth, and to the scientists who helped us learn more about mass spec in a week than we thought possible. See you next year at ASMS!

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At PacBio’s UGM, Top-Tier Science and Praise for BluePippin

It was a pleasure to co-sponsor the Pacific Biosciences user group meeting in Baltimore this week. Based on our participation at the same event last year, we had high expectations for it — but even these were surpassed by the quality of speakers, number of attendees, and excellent presentations.

Naturally, the vast majority of talks focused on using the uniquely long reads generated by Single Molecule, Real-Time (SMRT®) Sequencing to do all sorts of interesting projects that can’t be accomplished with short reads. Luke Tallon from the University of Maryland and Adam Phillippy from the National Biodefense Analysis and Countermeasures Center spoke about microbial genomics, Cold Spring Harbor’s Dick McCombie presented work in yeast and plants, and human genome studies were reported by Ali Bashir at the Icahn Institute for Genomics and Multiscale Biology and Tina Graves-Lindsay at Washington University. Other speakers covered more recent applications of SMRT Sequencing, such as metagenomics, full-length isoform sequencing, methylation analysis, and more.

At this meeting last year, we were delighted to have a couple of speakers mention our automated BluePippin size selection tool, which can be used with the PacBio platform to filter out smaller DNA fragments; this allows the sequencer to focus on long fragments, increasing efficiency and average read length. This year, it was a tremendous honor to find that nearly every speaker mentioned using BluePippin. We had that and the new SageELF whole-sample fractionation tool on display at the user group meeting, and we were bowled over by how many attendees came by to check them out. This group kept us on our toes!

It was also great to hear that BluePippin users are already anticipating how they might use the SageELF in their SMRT Sequencing workflows. Some speakers mentioned the possibility that whole-sample fractionation could be used to capture plasmids and other small parts of an organism’s accessory genome that could otherwise be filtered out by the high-pass protocol. We’re eager to see how that and other applications could make these remarkable research efforts even better.

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