Pippin Platform Recommended for Nextera Mate Pair Size Selection
Illumina released new sample prep protocol guidelines for generating mate pair libraries with its Nextera kit, and we’re pleased to report that the Pippin platform is the recommended choice for automated size selection.
You can check out the Nextera Mate Pair Sample Preparation Guide here . (We’re under Size Selection in Chapter 3, beginning on page 40 of the Guide.)
Illumina says that using an extra size selection step offers “more stringent” sizing than AMPure alone and lets users make libraries with larger fragments and more precise distribution than a gel-free approach. While the company has validated a manual approach in addition to the Pippin platform, Illumina’s guidelines note that “in our experience running a standard agarose gel does not provide as robust and reproducible results as the Sage Pippin Prep.”
In the user document, Illumina recommends the Pippin Prep with the 0.75% cassette and “eluting fragments with a broad range of sizes, of 3 to 6 kb in width, increasing in width with increasing fragment length (e.g. 2–5 kb, 4–8 kb or 6–12 kb).”
For current Pippin users, we would like to add that you can also use the 0.75% agarose dye-free cassette (BLF7510) with the BluePippin for equivalent results.
Poster: Exome Sequencing with Ion Torrent and Pippin Prep
In a poster from Ion Torrent (Life Technologies) for 2012’s ASHG, scientists looked at exome sequencing by studying a familial trio on both the PGM™ and the Proton™ instruments. Size selection for both sequencers was performed on the Pippin Prep from Sage Science.
Using an enrichment process targeting protein-coding exons from various genetic databases (including GenCode, RefSeq, Ensembl, and others), the scientists report “an on-target read mapping rate of 80%.” The sequencing, which took about four hours on either instrument, generated more than 5 Gb of aligned sequence, with average depth greater than 50x.
The Life Technologies authors note that they used Pippin size selection for both instruments, selecting an average length of 200 bp for the Proton and 300 bp for the PGM. (Check out figure 3 of the poster to see the nice clean peak they generated with Pippin.) This step was followed by exome enrichment and amplification prior to sequencing. The results indicate that sequencing exomes on the Proton is better than five times more efficient than on the PGM, and the authors say that stat is expected to improve even more.
Overall, the authors say, this study demonstrates that “the combination of focused exome enrichment and Ion Torrent Systems-based sequencing and analysis provides an efficient, accurate, and rapid means to detect genetic variation in the well-annotated portion of the genome for state-of-the-art genetic disease research.”
Check out the poster here.
Tag Team: At DFCI, Pippin and Nextera Make Better Libraries
Over at the Dana-Farber Cancer Institute, scientists have been doing some really interesting work pairing the Pippin Prep with Illumina’s Nextera kit for library preparation.
Zach Herbert, associate director of the Molecular Biology Core Facilites at DFCI, says that he incorporated Pippin size selection when it became clear that the genomic libraries generated by Nextera could use some additional optimization. “It’s really beneficial to have a narrower size range than the Nextera kit generates on its own,” he says.
Herbert uses the efficient Nextera for much of the small genome work and some of the larger amplicon projects that are sent to his core lab. In order to optimize reproducibility, flow cell clustering on the MiSeq, and downstream analysis, Herbert added a Pippin step to generate very tightly sized libraries after the Nextera tagmentation protocol.
The Pippin/Nextera tag team also shows value beyond de novo assemblies. “Having a narrow and known size distribution makes calculating the molarity a lot easier so you can get a better cluster density and maximize the number of reads,” Herbert says. It’s also a boon for pooling samples. Attempting to pool samples with a broad size range in equimolar amounts is very tricky — “but if all those libraries are the same size, then we’re much more likely to get an even distribution of that pool.”
To learn more about Herbert’s work pairing Nextera and Pippin, read our case study here.
DNA for Species Identification: A Study of Rodents
Carl Linnaeus would be proud: A recent paper in PLoS One demonstrates the use of next-gen sequencing with genetic barcodes to accurately identify more than 100 different species from the Rodentia order. In the study, amplicons were run on the Pippin Prep from Sage Science to remove non-specific PCR products.
“Next-Generation Sequencing for Rodent Barcoding: Species Identification from Fresh, Degraded and Environmental Samples,” the paper from Maxime Galan, Marie Pagès, and Jean-François Cosson at the Center for Biology and Management of Populations at INRA, uses 454 GS-FLX sequencing. The authors note that correct species assignment in the diverse Rodentia order is quite challenging with morphological data alone.
In this work, the authors selected a 136 bp fragment from cytochrome b as a mini-barcode and then used it on more than 900 samples to determine its utility in accurately identifying species. Following a validation step, hundreds of samples of unknown identity were analyzed and the mini-barcode worked about 85 percent of the time, the scientists report. They also successfully tested degraded rodent DNA samples, including museum specimens and feces from rodent-eating predators.
The authors conclude, “This study demonstrates how this molecular identification method combined with high-throughput sequencing can open new realms of possibilities in achieving fast, accurate and inexpensive species identification.”
Holiday Greetings from Sage Science
It’s that time of year again — we slow down at work, do some reflection, and gear up for a great (lucky?) ‘13….
In the field of genomics, 2012 was a fascinating year. On the NGS front, placements of desktop sequencers took off in terms popularity and performance, and the industry closed in on the $1000 genome. On the research front, studies on gene regulation continues to boggle the mind, and there seems to be an up-tick in interest in structural variation. This, and advances in cancer genomics, could very likely set off an avalanche of whole genome sequencing over the next few years.
For Sage Science, 2012 was a big year as well. We’ve established the Pippin as the go-to platform for high quality genomic libraries. And we launched BluePippin, the second in our Pippin automated size selection line, which features pulsed-field electrophoresis and collects fragments up to 50 Kb.
We were also delighted to see so many publications this year from scientists using the Pippin platform. Here are a few of our favorites:
• “Double Digest RADseq: An Inexpensive Method for De NovoSNP Discovery and Genotyping in Model and Non-Model
Species” from Peterson et al. in PLoS One
• “Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method” from Duhaime et al. in Environmental Microbiology
• “Transcriptional Amplification in Tumor Cells with Elevated c-Myc” from Lin et al. in Cell
• “Synthetic Spike-in Standards Improve Run-Specific Systematic Error Analysis for DNA and RNA Sequencing” from Zook et al. in PLoS One
With that, we wish everyone a happy holiday season! See you in 2013.