National Aeronautics and Space Administration Genetic Inventory Task Final Report Volume 1 of 2 Kasthuri Venkateswaran Myron T. La Duc Parag Vaishampayan OTHER SIGNIFICANT CONTRIBUTORS Shariff Osman Kelly Kwan Emilee Bargoma Alexander Probst Moogega Cooper James N. Benardini James A. Spry September 2012 National Aeronautics and Space Administration Jet Propulsion Laboratory California Institute of Technology Pasadena, California www.nasa.gov JPL Publication 12-12 09/12 JPL Publication 12-12 Genetic Inventory Task: Final Report Copyright This research was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement by the United States Government or the Jet Propulsion Laboratory, California Institute of Technology. © 2012 by the California Institute of Technology. U.S. Government sponsorship acknowledged. This report shall be referenced as follows. For Volume 1: Venkateswaran, K., La Duc, M. T., and Vaishampayan, P. (2012) Genetic Inventory Task: Final Report, Volume 1, JPL Publication 12-12. Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA. For Volume 2: Venkateswaran, K., La Duc, M. T., and Vaishampayan, P. (2012) Genetic Inventory Task: Final Report, Volume 2, JPL Publication 12-12. Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA. i JPL Publication 12-12 Genetic Inventory Task: Final Report Contents CONTENTS VOLUME 1 Abstract ......................................................................................................................................................................... vi Executive Summary ..................................................................................................................................................... vii 1. Introduction ........................................................................................................................................................ 1-1 2. Strategic Perspective ......................................................................................................................................... 2-1 3. Objectives and Expected Impact ........................................................................................................................ 3-1 3.1 Definition of Genetic Inventory ................................................................................................................ 3-1 3.2 Objectives ................................................................................................................................................ 3-1 3.3 Emphasis ................................................................................................................................................. 3-1 3.4 Expected Impact to Present Knowledge .................................................................................................. 3-3 4. Genetic Inventory Study Methodology ............................................................................................................... 4-1 4.1 General Approach ................................................................................................................................... 4-1 4.2 Study Methodology to Collect, Process, and Archive Nucleic Acids ....................................................... 4-2 4.3 Study Methodology to Assess the Phylogenetic Breadth of Microorganisms .......................................... 4-2 5. Sample Collection from Low-Biomass Surfaces ................................................................................................ 5-1 5.1 Objectives ................................................................................................................................................ 5-1 5.2 General Approach ................................................................................................................................... 5-2 5.3 Sampling Device Selection ...................................................................................................................... 5-2 5.4 Laboratory Model–Based Study .............................................................................................................. 5-4 5.5 Summary of Sample Collection from Low-Biomass Surfaces ............................................................... 5-10 5.6 Key Considerations ............................................................................................................................... 5-11 6. Sample Processing ............................................................................................................................................ 6-1 6.1 Objectives ................................................................................................................................................ 6-1 6.2 General Approach ................................................................................................................................... 6-2 6.3 Pooling of Low-Biomass Sample Volumes .............................................................................................. 6-3 6.4 Concentration of Sample DNA ................................................................................................................ 6-3 6.5 Bead-Beating prior to DNA Purification ................................................................................................... 6-4 6.6 DNA Purification Methods ....................................................................................................................... 6-4 6.7 Results of Sample Concentration ............................................................................................................ 6-6 6.8 Results of DNA Purification ..................................................................................................................... 6-6 6.9 Key Considerations ............................................................................................................................... 6-10 7. Sample Analysis................................................................................................................................................. 7-1 7.1 Objectives ................................................................................................................................................ 7-1 7.2 General Approach ................................................................................................................................... 7-1 7.3 Quantitative-PCR Analysis ...................................................................................................................... 7-2 7.4 Species-Specific Quantitative-PCR Analysis ........................................................................................... 7-2 7.5 16S rDNA Cloning and Sanger Sequencing Method ............................................................................... 7-2 7.6 PhyloChip Microarray Analysis ................................................................................................................ 7-3 7.7 Titanium 454 FLX Tag-Encoded Pyrosequencing ................................................................................... 7-4 7.8 Controls and Lower Detection Limits of Assays Employed ..................................................................... 7-5 7.9 Key Considerations ................................................................................................................................. 7-5 ii JPL Publication 12-12 Genetic Inventory Task: Final Report Contents 8. Implementation of a DNA Archiving System for the Genetic Inventory Task ..................................................... 8-1 8.1 Objectives ................................................................................................................................................ 8-1 8.2 General Approach ................................................................................................................................... 8-2 8.3 Controlled Laboratory–Based Study ........................................................................................................ 8-2 8.4 Environmental-Sample-Based Study ....................................................................................................... 8-5 8.5 Key Findings for Archiving Samples ........................................................................................................ 8-6 8.6 Perceived Impact and Genetic Inventory–Deployed DNA Storage Practices .......................................... 8-7 8.7 Sample Information Management System (SIMS) .................................................................................. 8-7 8.8 Summary of DNA Archiving Implementation for the Genetic Inventory Task .......................................... 8-8 9. Bioinformatic Approaches to Quality Assurance Assessment of Raw Data ....................................................... 9-1 9.1 Objectives ................................................................................................................................................ 9-1 9.2 Available Computational Tools for Assessing Microbial Diversity ........................................................... 9-2 9.3 Implementation of Bioinformatic and Biostatistical