Conference Program - ABRF 2011 - Association of Biomolecular ...

uniformity, reproducibility of enrichment, and ability to detect SNPs,insertion/deletions, and CNVs across Illumina (Genome Analyzer IIxand HiSeq2000) and SOLiD platforms. We highlight the utility of theSureSelect All Exon product portfolio for a wide variety of applicationsprimarily due to the high specificity and excellent cross-platformsequence coverage. SureSelect All Exon designs also provide a meansfor standardization, consistency of performance, and reliability acrossmultiple laboratories.152 PCR-Free Nextera Di-TaggedDNA Library Preparation for NGSApplicationsC. Kinross, H. Grunenwald, B. Baas, I. Goryshin,N. Caruccio, M. MaffittEpicentre Biotechnologies, Madison, WI, UnitedStatesThe Nextera TM technology for generating libraries of di-tagged DNAfragments is rapidly becoming the preferred method for massivelyparallel DNA sequencing. Despite rapid advances in sequencinginstrument throughput, classic library preparation by step-wise ligationof adaptors is a time-intensive and throughput-limiting bottleneck.PCR amplification of libraries prior to cluster generation is also amajor concern because of its possibility to reduce library complexity,particularly in regions of extreme G+C content (high or low), therebyproducing uneven genome coverage and confounding mapping andassembly. In this study, we describe novel modifications of the Nextera TMlibrary preparation system to address such library preparation biasby eliminating PCR amplification. Sequencer-ready libraries can beobtained from as little as 200 ng of genomic DNA in 3 hours with90-minutes of hands-on time. Deep sequencing of genomic librariesindicates that this system reduces coverage bias and GC bias, as well asimproves library diversity.153 Semiconductor Sequencing for LifeJ. Myers, J. RothbergIon Torrent, South San Francisco, CA, United StatesIon Torrent has invented the first device-a new semiconductor chipcapableof directly translating chemical signals into digital information.The first application of this technology is sequencing DNA. The deviceleverages decades of semiconductor technology advances, and in justa few years has brought the entire design, fabrication and supply chaininfrastructure of that industry-a trillion dollar investment-to bear on thechallenge of sequencing. The result is Ion semiconductor sequencing,the first commercial sequencing technology that does not use light, andas a result delivers unprecedented speed, scalability and low cost. Allof these benefits are a result of applying a technology that is massivelyscalable, as proven by Moore’s Law, to a task that has traditionally usedoptics-based solutions, which work in a linear fashion: increasing capacityrequires increasing the number of signals that must be read resultingin longer run times, higher capital costs and ever more sophisticatedoptics. By contrast, Ion Torrent semiconductor technology can provideincreases in chip capacity without impacting capital costs or runtime.Ion Torrent sequencing uses only natural (label-free) reagents andtakes place in Ion semiconductor microchips that contain sensors whichhave been fabricated as individual electronic detectors, allowing onesequence read per sensor. We will show how the technology has scaledin just a few months from ~1 million sensors in the first-generationIon 314 chips to ~7 million sensors in the second-generation Ion 316chips-all while maintaining the same 1- to 2-hour runtime. We will alsodemonstrate that Ion semiconductor sequencing provides exceptionalaccuracy, long read length and scalability on a single, affordable benchtopsequencing platform.154 A Comparison of Post-DNASequencing Dye-Terminator RemovalProtocolsM. Zianni, A. McCoyThe Ohio State University, Columbus, OH, UnitedStatesCapillary electrophoresis, a method for separation of ions based upontheir size to charge ratio, remains in high demand for DNA sequencing.In the process of dideoxynucleotide terminator sequencing,unincorporated nucleotides and other contaminants remaining in thereaction mixture can cause multiple issues in the electropherograms,such as unincorporated dye peaks, missed base calls, decreased signalstrength or a complete lack of data as a result of blocked capillaries. Avariety of dye-terminator removal protocols exists to clean and purifythe sequencing reaction extension products. To determine their qualityand reproducibility, six protocols were tested with one large volumecontrol reaction aliquoted into a 96-well PCR plate. The protocolsincluded ethanol precipitation, gel filtration, and 4 solid phasereversible immobilization procedures with 1 utilizing the surface of aplate and the other 3 utilizing magnetic beads. The cleaned and purifiedsequencing reactions were processed on the 3730 DNA Analyzer(Applied Biosystems), and the contiguous read lengths, QV20+ scores,and signal strengths of the resulting sequences were analyzed withSequence Scanner v1.0 (Applied Biosystems). Based on the results fromtwo replicate rounds of testing, the gel-filtration protocol provided thelongest contiguous read lengths and highest QV20+ scores.155 Simplified Reagents and Workflowsfor Robust Sample Preparation ofDNA, mRNA, and Small RNAF.J. Stewart, C.L. Hendrickson, L.M. Apone,D.B. Munafo, C.R. MeyerNew England Biolabs, Inc., Ipswich, MA, UnitedStatesAs yields of data generated by the Illumina, SOLiD, and 454sequencing platforms increase, NGS users have transitioned fromperforming multiple sequencing runs per sample to multiple samplesper sequencing run. As a result, the bottleneck in sequencing labs hastransitioned from data generation to sample preparation, necessitatingthe development of streamlined library construction workflows. Wehave developed a series of reagents to facilitate the easy preparationof numerous samples in parallel, compatible with both manual andautomated pipelines. These reagents reduce the amount of laborrequired, minimize error in reaction set up, and increase the stability ofenzymes used in the construction of libraries for DNA, mRNA and SmallRNA sequencing. As a result, these reagents enable the developmentof robust workflows for both individual and high throughput samplepreparation.Poster AbstractsABRF 2011 — Technologies to Enable Personalized Medicine • 75