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

Research GroupPresentation Abstracts(R3b) DNA Sequencing Research Group (DSRG)(R3b-1) Comparison of Custom Target EnrichmentMethods; Agilent vs. NimblegenA. Perera 4 , K. Bodi 1 , P.S. Adams 2 , D. Bintzler 3 , K. Dewar 5 ,D.S. Grove 6 , J. Kieleczawa 7 , R.H. Lyons 8 , T. Neubert 9 ,A.C. Noll 1 , S. Singh 10 , R. Steen 11 , M. Zianni 121Tufts University, Boston, MA, United States; 2 TrudeauInstitute, Saranac Lake, NY, United States; 3 DNA Analysis,Inc., Cincinnati, OH, United States; 4 Stowers Institute, KansasCity, MO, United States; 5 McGill University, Montreal, QC,Canada; 6 Pennsylvania State University, University Park,PA, United States; 7 Pfizer Research, Cambridge, MA, UnitedStates; 8 University of Michigan, Ann Arbor, MI, UnitedStates; 9 New York University, New York, NY, United States;10University of Minnesota, Minneapolis, MN, United States;11Harvard Medical School, Cambridge, MA, United States;12Ohio State University, Columbus, OH, United StatesOver the last four years, we witnessed the tremendous advances inNext Generation Sequencing (NGS) that have dramatically decreasedthe cost of whole genome sequencing. However, the cost of sequencinglarger genomes is still significant. In addition and depending on thegoal of study, whole genome sequencing creates a large amount ofadditional/auxiliary data that complicates data analysis. There areseveral commercial methods available for isolating subsets of genomesthat greatly enhance the efficiency of NGS by allowing researchersto focus on their regions of interest. For the 2009-11 DSRG study,we compared products from two leading companies; Agilent andNimblegen that offer custom enrichment methods. Both companiesobtained the same genomic DNA stock and performed DNA captureon the same specified regions. Following capture, the Illumina GenomeAnalyzer IIx system was used, in two different laboratories, to generatethe sequence data. We present our data comparing in terms of cost,quality, reproducibility and most importantly completeness and depthof coverage. Acknowledgements: We would like to thank Agilent,Illumina and Nimblegen for all their support in making this studypossible.(R3b-2) A Methodology Study for MetagenomicsUsing Next Generation SequencersS. Singh 1 , D. Grove 21Biomedical Genomics Center, University of Minnesota,Saint Paul, MN, United States; 2 Genomics Core Facility, HuckInstitutes for Life Sciences, Pennsylvania State University,University Park, PA, United StatesMetagenomics is one of several genomics applications, which hasbenefited immensely from the high throughput and cost efficacy ofNext generation sequencers. And although hundreds of studies onmetagenome analysis have been published over the past few years,the methodology for conducting them is still very much evolving.In this DSRG study we will evaluate the influence of various samplepreparation methods, specifically DNA extraction and amplificationapproaches, on data output along with a comparative analysis of Nextgeneration sequencing platforms. We will study the effect of thesedifferent experimental and technical strategies on determination ofsample biodiversity.(R4) Joint Session: Protein Sequencing ResearchGroup (PSRG) & Glycoprotein Research Group(gPRG)(R4a) PSRG 2011 Study Results: SensitivityAssessment of Terminal Sequencing TechniquesUsing an Unknown ProteinJ.J. Walters 1 , W. Sandoval 2 , K. Mawuenyega 3 ,J.S. Smith 4 , H. Remmer 5 , B. Xiang 6 , D. Suckau 7 , V. Katta 2 ,P. Hunziker 81Sigma-Aldrich, St. Louis, MO, United States; 2 Genentech,Inc., South San Francisco, CA, United States, 3 WashingtonUniversity School of Medicine, St. Louis, MO, United States,4University of Texas Medical Branch, Galveston, TX, UnitedStates, 5 University of Michigan, Ann Arbor, MI, UnitedStated, 6 Monsanto Company, St. Louis, MO, United States,7Bruker Daltonics, Bremen, Germany, 8 University of Zurich,Zurich, SwitzerlandEstablishing the N-terminal sequence of intact proteins plays a criticalrole in biochemistry and potential drug development. N-terminalsequence analysis is necessary for quality control of protein biologics,for determining sites of signal peptide cleavage events, as a first stepin elucidating the sequences of genes from uncommon species andfor the characterization of monoclonal antibodies. Automated Edmandegradation has been the method of choice for these analyses. However,alternate methods for N-terminal sequence analysis have emerged. Therecent PSRG studies have established that Edman sequencing and massspectrometry based techniques have varied strengths and weaknessesdepending on several experimental factors and both play an importantrole in terminal sequencing. With this complimentary role realized,the 2011 PSRG study attempts to evaluate the sensitivity limits of thevarious sequencing techniques. The PSRG distributed three samplesets of 3 tubes each, varying by sample format (lyophilized, gel sliceor membrane piece). Each set of three samples contains the samerecombinant protein with increasing amounts of material. The sequenceof this protein is not listed in any database. Participants could requestany one, two, or all three sample sets. Including PSRG committee, a totalof 38 participants requested 74 sample sets. The participants wereasked to determine as many amino acids from both termini by theirmethod of choice, and were encouraged to try multiple methods forsequence elucidation. Study participants were directed to a websiteto anonymously upload sequences and supporting data. Our analysisfocuses on the length and accuracy of the sequence calls reported bythe participants, and how that compares with decreasing amounts ofprotein and the type of sample format analyzed. A comparison of theresults obtained by Edman chemistry and by alternative technologiesas well as information on the type of instruments and protocols isreported.56 • ABRF 2011 — Technologies to Enable Personalized Medicine