At the University of Akron, geology and biology professor Hazel Barton shows her students the ropes. Literally. A veteran caver, Barton combined her passion for exploring some of the most remote locations in the world with her scientific interest in microbiology and became a widely acclaimed expert on the communities of extremophiles living in caves.
Here in the hallways of Sage Science headquarters, Barton is famous for another reason: she is the 1,000th customer of our Pippin automated DNA size selection platform. As we celebrate this milestone, we wanted to share a quick profile of Barton’s science here on our blog.
Barton went on her first cave expedition on a class trip at age 14, and has been caving ever since. She never expected this interest to mix with her career, until her postdoc advisor and 16S sequencing pioneer Norman Pace pointed out that she could get to places on Earth that most people couldn’t, giving her a remarkable opportunity to study microbiology in unexplored realms. “Rather than my hobby being something that was a distraction from my science, all the years of experience I had in caves allowed me to go in and recognize things that were unusual and that could be biological,” she says. Today, her research sites include caves in Kentucky, New Mexico, Venezuela, Brazil, Belgium, and China.
When Barton first began her explorations with microbiology in mind, conventional wisdom suggested that the physical isolation and lack of sunlight characteristic of caves would mean they couldn’t support much life. Barton’s early research demonstrated that there was actually “a profound level of diversity,” she says. “I spent the rest of my career trying to figure out why caves are so diverse and what are the community interactions that support that diversity.” She has unearthed insights into relationships that allow these extremophiles to survive, from the microbes that work together to break down scarce nutrients to share an energy source to the “cheaters” that eat bacteria and even out population levels.
For Barton and the students who accompany her into the depths of these caves — a favorite sampling site is some 1,500 feet underground — extracting DNA is no simple task. “These microbes are ingrained in the rock,” she says. “It’s very hard to study them.” A gram of soil, for instance, might contain four to five orders of magnitude more bacteria than a gram of cave sediment. To make things even more challenging, less than 0.1% of those microbes can be cultured back in the lab.
That’s why Barton, whose team specializes in generating libraries from low-biomass samples, turned to the Pippin Prep. Her lab had been finding it difficult to get enough product for 454 sequencing, but scientists at a collaborating center in Kentucky “would send us these beautiful images of these gels” that they had generated from the same sample. “They could effectively get 4 nanograms of product and we would just lose it all when we would gel purify,” says Barton. When she asked why, she learned that the center’s success was due to Pippin.
She brought Pippin Prep into her own lab for testing and decided it was a no-brainer to purchase the instrument. “For the first time in a long time, we were able to clone products that we hadn’t been able to clone before because there just hadn’t been enough DNA,” Barton says. “The Pippin is so valuable to us because it’s all about very low biomass and getting a high percentage recovery.”
Many thanks to Barton and all the amazing Sage Science customers who helped us get to this point. Here’s to the next thousand!
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 small RNAs.
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!
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:
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!
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!