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. (2017) IEEE Aerospace (In Press)

Detect EsHmated TranslocaHon DNA Analysis Events Sequence E. coli CsgG Neural Network-based Data Processing 5/3/17 Carr – FISO – Life Detection 32 Non-standard Bases?

Extra-terrestrial nucleobases Guanine (G) is a standard nucleobase. idenHﬁed in meteorites, e.g. Hypoxanthine is next most abundant. Callahan et al. (2011) Hypoxanthine + ribose = Inosine Guanine Hypoxanthine Xanthine + = Purine 2,6-Diaminopurine CC BY-SA 3.0 h-ps://goo.gl/SGBkXz

Our approach to detec@ng inosine: 1) Use synthe@c DNA polymer made of CICICICICIC… poly(dI-dC) 2) Consider that CsgG pore current largely reﬂects about 3 bases.

5/3/17 Carr – FISO – Life Detection 33 Detection of Inosine Nucleoside Using Strand Sequencing

Lambda: many 3-mers

24 events in 23.8 s (1.01 events/s) Poly(dI-dC): 3-mers: CIC or ICI

Carr et al. IEEE Aerospace (2017) In Press

5/3/17 Carr – FISO – Life Detection 34 Strand Sequencing: Successes and Challenges • DNA • Input DNA • RNA 1000 ng nominal vs. 2 pg • Non-standard requirement (500,000X) vs. bases (some) data requirement (5000X) • Non-standard • Yield polymers Currently, get 1-4 bases per Solid state 1M bases into library prep sequencing?

Failure modes tested to date: 1) Heat (protein denaturaHon), 2) No input DNA: Hachey et al. AbSciCon (2017) Abstract # 3454.

5/3/17 Carr – FISO – Life Detection 35 A Common Tree of Life on Earth and Mars?

Mars Life?

Any Mars life should be deeply branching, isolated for ~3.5 Gy. Credit: CC BY 3.0 h-ps://goo.gl/9BYVui

5/3/17 Carr – FISO – Life Detection 36 Advantages & Disadvantages

• Unambiguous signature of life • Biological reagent preservaHon hard • IdenHfy Earth ContaminaHon • Biological approach: assumpHons • Amazing theoreHcal sensiHvity (~10-21) about type of nucleic acids • Can detect a DNA, RNA (nascent), non- • Currently, pracHcal sensiHvity limits standard bases

5/3/17 Carr – FISO – Life Detection NASA/JPL/Ted Stryk/Space Science37 Institute 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 38 SETG Technology Readiness Level (TRL) Evolution

TRL 3 TRL 4 Pre-TRL 5 Pre-TRL 6

Extraction modules Extraction

Manual Automated Automated* Syringe Pump Electrolytic Pumps Fluid handling Stepper + Valve Stepper + Valve Nanopore TRL 4 Pre-TRL 5 Sequencing

Data Processing + thermal insulation + control pressure vessel (base) Sequencing

Mostly Automated Portable Sequencing, Section view of Pre-TRL 6 model Manual loading Available Today with realistic volume allocations

Carr et al. 2017 IEEE Aerospace (In Press)

5/3/17 Carr – FISO – Life Detection 39 Space Validation of Sequencing

Credit: NASA

5/3/17 Carr – FISO – Life Detection 40 Preview: Sequencing on “Mars” April 5, 2017

Carr et al. (in prep)

h-ps://twi-er.com/carr_lab/status/849732444576256002

5/3/17 Carr – FISO – Life Detection 41 Challenges for Life Detection Mars Enceladus Europa

Special Regions Plume Sampling Radiation Sample Acquisition Autonomous Operations Planetary Protection XNA Sequencing Credit: NASA/JPL NASA/JPL Credit:

5/3/17 Carr – FISO – Life Detection 42 A Biological Future

Clinical Medicine Search for Life SyntheHc Biology Planetary ProtecHon

Environmental In-situ Monitoring Human Health Resource In Space UHlizaHon

Food Medicine Life Support Credit: NASA Manufacturing

5/3/17 Carr – FISO – Life Detection 43 Credits: NASA Space Tourism Board

Thank you! Special thanks to FISO organizers, Zuber & Ruvkun labs, SETG alumni.

Maturation of Instruments for Solar System Exploration: NNX15AF85G (MATISSE) Astrobiology Instrument Development: NNX09A076G, NNX08AX15G (ASTID) Field science & instrument testing: NNX09AO76G, NNX12AM83G (MMAMA)

[email protected] | setg.mit.edu | @carr_lab

5/3/17 Carr – FISO – Life Detection 44 Questions?

Rover Data Processing Sequence Analysis

Icy Moon Proteobacteria Orbiter Firmicutes DNA/RNA Biologically XNA Archaea Ocean -based Bacteroidetes extraction Nanopore Explorer Chloroﬂexi Sequencing

ValidaHon using hard to lyse Non-standard bases RadiaHon Resistant spores (Bacillus sub3lis) (Inosine nucleoside) Neural Network-based Memory (CBRAM) Data Processing

[email protected] | setg.mit.edu | @carr_lab

5/3/17 Carr – FISO – Life Detection 45 Selected References (2017)

• Full list: http://setg.mit.edu/publications/ • Carr CE, Mojarro A, Hachey J, Saboda K, Tani J, Bhattaru SA, Smith A, Pontefract A, Zuber MT, Finney M, Doebler R, Brown M, Talbot R, Nguyen V, Bailey R, Ferguson T, Church G, Ruvkun G. Towards In Situ Sequencing for Life Detection. Aerospace Conference, 2017 IEEE. March 4-11, Big Sky, Montana. Session 2.07 In Situ Instruments for Landed Surface Exploration, Orbiters and Flybys. Paper # 2353 (In Press) Author’s manuscript: https://goo.gl/aCcsx0 • Carr CE, A. Mojarro, J. Hachey, A. Pontefract, R. Doebler, M. Brown, G. Ruvkun, and M. T. Zuber. Progress and Challenges for Life Detection via Nucleic Acid Sequencing. Astrobiology Science Conference, Mesa, Arizona, April 24–28, 2017. Abstract #3395 http://www.hou.usra.edu/meetings/abscicon2017/pdf/3395.pdf • J. Hachey, A. Pontefract, M. T. Zuber, G. Ruvkun, C. E. Carr. Sequencing Nothing: Exploring Failure Modes of Nanopore Sensing and Implications for Life Detection. Astrobiology Science Conference, Mesa, Arizona, April 24–28, 2017. Abstract # 3454 http://www.hou.usra.edu/meetings/abscicon2017/pdf/3454.pdf • A. Mojarro, J. Hachey, R. Bailey, M. Brown, R. Doebler, G. Ruvkun, M. T. Zuber, C. E. Carr. Nucleic Acid Extraction and Sequencing from Low-Biomass Synthetic Mars Analog Soils. Lunar & Planetary Sci XLVIII, The Woodlands, Texas, March 21-25, 2017. Abstract # 1585 http://www.hou.usra.edu/meetings/lpsc2017/pdf/ 1585.pdf • Pontefract, J. Hachey, A. Mojarro, V. K. Walker, H. Rowedder, T. F. Zhu, C. Lui, M. T. Zuber, G. Ruvkun, C. E. Carr. Understanding Habitability and Biosignature Preservation in a Hypersaline Mars Analog Environment: Lessons from Spotted Lake. Lunar & Planetary Sci XLVIII, The Woodlands, Texas, March 21-25, 2017. Abstract # 1124 http://www.hou.usra.edu/meetings/lpsc2017/pdf/1124.pdf • Tani J, Ruvkun G, Zuber MT, Carr CE. On Neuromorphic Architectures for Efﬁcient, Robust, and Adaptable Autonomy in Life Detection and Other Deep Space Missions. Planetary Science Vision 2050 Workshop, Washington, DC – Feb 27-Mar 1, 2017. Abstract # 8080 http://www.hou.usra.edu/meetings/V2050/pdf/ 8080.pdf

5/3/17 Carr – FISO – Life Detection 46