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Towards in Situ Sequencing for Life Detection FISO Seminar – May 3, 2017 Towards In Situ Sequencing for Life Detection Christopher E. Carr Research Scientist, MIT Research Fellow, MGH Science PI, Search for Extra-Terrestrial Genomes (SETG) [email protected] | setg.mit.edu | @carr_lab Image: Jenny Mo-ar/NASA Search for Extra-Terrestrial Genomes (SETG) Team Not pictured: Levon Avakian, John Cashion, SETG Alumni 5/3/17 Carr – FISO – Life Detection 2 “I believe we are going to have strong indica@ons of life beyond Earth in the next decade and defini@ve evidence in the next 10 to 20 years” – Ellen Stofan, (then) Chief Scien3st (NASA) April 7, 2015 5/3/17 Carr – FISO – Life Detection 3 Today • What is life? • Where should we search for it? • How should we detect it? • What comes next? 5/3/17 Carr – FISO – Life Detection 4 Today • What is life? • Where should we search for it? • How should we detect it? • What comes next? 5/3/17 Carr – FISO – Life Detection 5 Life As We Know It DNA RNA “RNA World” Proteins Proper@es Poten@al Features EvoluHon InformaHonal polymers NASA “A self-sustaining Growth Cell and populaHon growth chemical system capable ReproducHon Cell division of Darwinian evoluHon” Metabolism Metabolites 5/3/17 Carr – FISO – Life Detection 6 Origin(s) of Life Astrochemistry ArHficial Life UV-driven synthesis Volcanism Hydrothermal Vents Mural at NASA Ames Research Center 5/3/17 Carr – FISO – Life Detection 7 Requirements for Life (as we know it) • CHNOPS elements • Liquid water / water activity > 0.61 • Redox gradient (energy flux) • Moderate temperatures • pH, salinity, pressure, etc. What environments meet the requirements? NAP h-ps://goo.gl/110XN5 5/3/17 Carr – FISO – Life Detection 8 Today • What is life? • Where should we search for it? • How should we detect it? • What comes next? 5/3/17 Carr – FISO – Life Detection 9 Searching for Life Beyond Earth Other Exoplanets Ocean Worlds… Mars A direct search for life Enceladus James Webb Space Telescope Europa Credits: NASA/JPL-Caltech/SETI 5/3/17 Carr – FISO – Life Detection 10 Is Carbon Based Life Universal? “Weak Panspermia” Nucleobases Sugars (Ribose) Comet (simulated) Meteorite(s) ESA/Rose-a/NAVCAM, CC BY-SA IGO 3.0 CC BY-SA 3.0 h-ps://goo.gl/SGBkXz Synthesis of prebio3c molecules in early solar nebula (Nuevo et al. 2009, 2012; Ciesla & Sandford, 2012), ribose and other sugars in late solar nebula (Meinert et al. 2016); Meteori3c amino acids & nucleobases (Engel et al. 1997; Mar3ns et al. 2008; SchmiL-Kopplin et al. 2010; Cooper et al. 2011; Callahan et al. 2011) 5/3/17 Carr – FISO – Life Detection 11 Shared Ancestry? ~4 billion years ago … “Lithopanspermia” Calcula3on/Simula3on (Gladman & Burns, 1996; Gladman et al. 1996; Gladman et al. 1997; Mileikowsky et al. 2000); Low temperature meteori3c transfer (Weiss et al. 2000); Microbes survive ejec3on shock (Burchell et al. 2004; Stöffler et al. 2007; Horneck et al. 2008; Meyer et al. 2011) Credit: ESO/M. Kornmesser CC 4.0 h-ps://goo.gl/7Vz5eS 5/3/17 Carr – FISO – Life Detection 12 Potential Refugia for Life on Mars Poten@al Near-Surface Zones of Extant Life Low Exposure Age Regions Recurring Slope Lineae Water Ice Fog / AcHve Fresh Impact Craters (RSLs) - Liquid Brines? Water Cycle? HiRISE NASA/JPL/ASU/MSSS HRSC/MEX/ESA Mars Odyssey / Mars Global Surveyor / NASA/JPL/ASU Subsurface Regions Example: Subsurface environments offer UV and radiaHon shielding, heat, moisture Extensive overlap between Mars and Earth of zones habitable for life as we know it (Jones et al., 2011) HiRISE NASA/JPL/ASU/MSSS Lava Tube h-p://goo.gl/GupK1H 5/3/17 Carr – FISO – Life Detection 13 5/3/17 Carr – FISO – Life Detection 14 5/3/17 Carr – FISO – Life Detection 15 How universal is biochemistry? 4.6 Ga 4.1 3.8 3.5 Gya Extant life? 0 Venus Comets Late Heavy Isotopic evidence Earth Bombardment: of life on Earth Meteoritic Start of transition from Mars transfer between wet to dry on Mars Titan Earth and Mars Meteorites (and Venus?) Complex organics Europa Fossil evidence Mars: Enceladus: of life on Earth Related life? 2nd genesis? form, mix in the Enceladus solar nebula Shadow biosphere on Earth? • Weak Panspermia: Common building blocks of life – Synthesis of prebiotic molecules in early solar nebula (Nuevo et al. 2009, 2012; Ciesla & Sandford, 2012), ribose and other sugars in late solar nebula (Meinert et al. 2016) – Meteoritic amino acids & nucleobases (Engel et al. 1997; Martins et al. 2008; Schmitt- Kopplin et al. 2010; Cooper et al. 2011; Callahan et al. 2011) • Lithopanspermia: Shared ancestry between Earth and Mars? – Calculation/Simulation (Gladman & Burns, 1996; Gladman et al. 1996; Gladman et al. 1997; Mileikowsky et al. 2000) – Low temperature meteoritic transfer (Weiss et al. 2000) – Microbes survive ejection shock (Burchell et al. 2004; Stöffler et al. 2007; Horneck et al. 2008; Meyer et al. 2011) 5/3/17 Carr – FISO – Life Detection 16 Today • What is life? • Where should we search for it? • How should we detect it? • What comes next? 5/3/17 Carr – FISO – Life Detection 17 Searching for Life Beyond Earth Proper@es of Life Biomarkers • Metabolism • Biofabrics • Growth • BiomineralizaHon • ReproducHon • Body fossils • EvoluHon • SpaHal chemical pa-erns • Biogenic gases (methane) • Isotope raHos e.g. Grotzinger et al. 2012 • Future missions: Biogenic organic molecules (amino acids, lipids, nucleic acids) • Need definiHve biomarkers! Charged linear informa7onal polymers e.g., Klein 1978; Klein 1979 likely universal for aqueous-based life. 5/3/17 Carr – FISO – Life Detection 18 Priority: Biogenic Organic Molecules On icy moons: biogenic organic molecules even more important, because some biosignatures are not present or are inaccessible. 5/3/17 Carr – FISO – Life Detection 19 5/3/17 Carr – FISO – Life Detection 20 Why nucleic acids? 5/3/17 Carr – FISO – Life Detection 21 Survival Time of DNA Model of DNA Hydrolysis Survival of the coldest adapted from Millar & Lambert, 2013 Mars Mars temperature preserves DNA on longer 3mescales versus Earth 5/3/17 Carr – FISO – Life Detection 22 Search for Extra-Terrestrial Genomes (SETG) Rover Data Processing Sequence Analysis Icy Moon Proteobacteria Orbiter Firmicutes DNA/RNA Biologically XNA Archaea Ocean -based Bacteroidetes extraction Nanopore Explorer Chloroflexi Sequencing Current TRL 4 ValidaHon using hard to lyse Non-standard bases RadiaHon Resistant spores (Bacillus sub3lis) (Inosine nucleoside) Neural Network-based Memory (CBRAM) Data Processing 5/3/17 Carr – FISO – Life Detection 23 Extraction Modules (4) Sequencing Volume-accurate (internal) Data Pre-TRL6 Processing (internal) SETG Model: Fluidics • 4 extracHon modules • 2 sequencers 9.5 cm 22 cm Carr et al. 2017 14cm IEEE Aerospace (In Press) Current Best Average System Budget Allocation Specification Estimate Contingency Margin System Volume 2.7 L 28% 3.4 L 4.3 L System Mass 3.7 kg 28% 4.8 kg 25% 6.0 kg Energy (Per Sample) 130 W-hr 31% 170 W-hr 210 W-hr 5/3/17 Carr – FISO – Life Detection 24 Abundance & Sensitivity 1 10 102 103 104 105 106 107 108 109 Cell Density (#/g) 5 Low-moisture Europa Ocean 2.5 10 Saturated · terrestrial energetic a bacterial analogs of upper limit? culture 1 ppb Mars (Atacama) DNA (mass/mass) 10-15 10-14 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 B. subtilis ATCC 6633 spores TRL6 Target 500 ng DNA for 50 mg sample Carr et al. (2017) AbSciCon Abstract #3395 5/3/17 Carr – FISO – Life Detection 25 Subsystem Requirements Sample Delivery Extraction Sequencing Analysis Forward Contamination? 1 ml/50 mg after any Putative concentration (Mars) Life? OmniLyse ® 4 10 spores 5% 0.06% Target >1M bases called 40 pg DNA Yield Yield Achieved: Detection of known Current 0.0001% (typical) B. subtilis spores in Earth organism with Best 0.0025% (optimal) Mars analog soils <30 ~kb length reads Carr et al. (2017) AbSciCon Abstract #3395 5/3/17 Carr – FISO – Life Detection 26 Mars Simulants MOLA Science Team Perchlorate Alkaline Acid Salt Basalt = Lunar analog Aeolian/JSC (global) Synthe3c samples enable controlled experiments Simulants derived from Schuerger et al. 2012 5/3/17 Carr – FISO – Life Detection 27 Extracting Nucleic Acids Semi-universal extrac3on protocol for all tested Mars analogs: • Sample: Mars analog sediment + B. sub3lis ATCC 6633 spores. Mojarro et al. • Pre-lysis desalHng. Astrobiology • Add compeHHve binders. (accepted) • Solid phase extracHon on beads. • No organic solvents! 18.5% 15.4% pump failure 8.21% 6.67% Mojarro et al. 5.64% LPSC XLVIII 1.9% 0% 0% 0% 0% (2017) #1585 NTC Blank Perchlorate Water DNA Yield wash DNA to ddPCR binding buffer DNA extraction desalt binding buffer waste elution spores competitive binders wash soil elution buffer elution sample waste 5/3/17 Carr – FISO – Life Detection 28 SpaceX CRS-8, April 8, 2016 4:43 pm EDT NASA WetLab-2 Julie Schonfeld RNA ExtracHon On-Orbit 5/3/17 Carr – FISO – Life DetectionJeff Williams Why | Where | Status | Case Study | Conclusions Credits: NASA 29 Images: NASA DNA Sequencing DNA Typical Extract Condition Sequence Microbial Cell Break Cell Convert DNA Read out sequence Membrane to readable "Library" of bases (A, C, G, T) (technology dependent) Separate DNA Typically short Can involve "cleaning", from Proteins, fragments (150-500 adding known ends, Lipids, etc. bases long) selecting certain gene Concentrate DNA regions, amplifying, etc. 5/3/17 Carr – FISO – Life Detection 30 Two Nanopore Sequencing Methods Sequencing by Strand Synthesis Sequencing T C A Tagged (pA) Nucleotides Current C Time (milliseconds) T G Monitor ionic current (I) A I through nanopore(s) I Estimate DNA bases using statistical models MinION Mk 1B 2048 pore array Carr et al. (2017) IEEE Aerospace (In Press) 5/3/17 Carr – FISO – Life Detection 31 Strand Sequencing of /sm5hPT Enterobacteria Phage Lambda goo.gl 48.5 kb genome, used as control for nanopore sequencing h-ps:// Duda Bob 42 events in 243 ms (170 events/s) Carr et al.
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