single molecule sensing and sequencing for life detection

Single Molecule Sensing and Sequencingfor Life Detection Beyond Earth

Christopher E. Carr1-2,*, Gary Ruvkun2,3, Maria T. Zuber1

1 Department of Earth, Atmospheric and Planetary Sciences, MIT 2 Department of Molecular Biology, MGH 3 Department of Genetics, Harvard Medical School* Research Scientist, MIT and Research Fellow, MGH; [email protected], cecarr.com, @carr_c_e

Image:JennyMottar/NASA

AstrobiologyScienceStrategyfortheSearchforLifeintheUniverse – April25,2018

?

Funding: Maturation of Instruments for Solar System Exploration (MATISSE), NNX15AF85GPrior support from NASA MMAMA, ASTID programs.

4/25/18 Carr – Astrobiology Science Strategy – Single Molecule Sensing & Sequencing

Notpictured:JonathanBorowsky,Levon Avakian,JohnCashion,SETGAlumni

2

Life As We Know It


PropertiesEvolutionGrowth

ReproductionMetabolism

PotentialFeaturesInformationalpolymersCellandpopulation growthCelldivisionMetabolites

NASA “Aself-sustainingchemicalsystemcapableofDarwinianevolution”

DNA

RNA

Proteins

“RNAWorld”

3

E.Coli280fg drymass139fg C(7billion atoms)

154fg protein50-60fg RNA4-16fg DNA25fg lipid

1femtogram (fg)=10-15 g

Why search for life as we know it?

4/25/18 Carr – Astrobiology Science Strategy – Single Molecule Sensing & Sequencing 4

Sugars(Ribose)

Comet

ESA/Rosetta/NAVCAMCCBY-SAIGO3.0

Meteorite(s)

https://goo.gl/SGBkXzCCBY-SA3.0

Nucleobases

GuanineAmino Acids

“WeakPanspermia” Common Physicochemistry?

Enceladus Europa

Nature(2001)https://goo.gl/Pw8nMM

Ventorigin(s) oflife?

Mars

CommonAncestry?

99% 1%Meteoritic Transfer

Surface orventorigin oflife onEarthorMars?


e.g.,Klein1978;Klein1979

PropertiesofLife• Metabolism• Growth• Reproduction• Evolution

Chargedlinearinformationalpolymerslikelyuniversalforaqueous-based life.

Biomarkers• Biofabrics• Biomineralization• Bodyfossils• Spatialchemicalpatterns• Biogenicgases(methane)• Isotoperatios• Futuremissions:Biogenicorganic

molecules(aminoacids,lipids,nucleicacids)

• Needdefinitivebiomarkers!

e.g.Grotzinger etal.2012

Searching for Life Beyond Earth

5

“Definitive” Biomarker Examples


Credit:EnceladusLifeFinder(ELF)team.

AminoAcidAbundanceDistributionDifferentfrom

ExpectedAbioticDistribution

Credit:EnceladusLifeFinder(ELF)team.

Repeatingsubunits&clusteringoflipids(membrane

buildingmolecules)

Longchargedlinearpolymersofappreciablelength,possibly

withconservedsequence

DNAorothernucleicacidsorrelatedpolymers

AminoAcidComplexity

Mars?

The Search for Extra-Terrestrial Genomes (SETG)


NucleicAcidIsolation Nanopore Sequencing DataAnalysis

Current TRL 4; system specification: ~6 kg, ~4 L, ~200 W-hr (with 25% system margin).

7

Mars Regolith

Abundance & Sensitivity


SETGTRL6Target500ngDNA

for50mgsample(yields1-10Gbases)

Carretal.(2017)AbSciCon Abstract#3395

8

Protein 40ppt

Lipids 6ppt

DNA 2ppt

Europa1mlsamplePre-concentrated1000X

Sequencingsensitivityneedstoimprove~103 foricymoonsevenwithpre-concentration.

109108107106105104103101Cell Density (#/g)

Saturatedbacterialculture

aLow-moisture terrestrial analogs ofMars (Atacama)

DNA (mass/mass)

10-510-610-710-810-910-1010-1110-1210-1410-15

1 ppb

Europa Oceanenergeticupper limit?

B. subtilis ATCC 6633 spores

102

2.5 · 105

10-1310-14

Adapted from Millar & Lambert, 2013Mars

ResidencetimeofDNA

• Hydrolysis:~106 y(Earth),~109 y(Mars)

• SpaceRadiation: Below2m(Mars)

• Selfradiation:~107 y(EarthorMars)

• Example: 7∗105 yoldbacteriaDNAinpermafrostJohnsonetal. 2007

Survival Time of DNA

MarstemperaturepreservesDNAonlongertimescales versusEarth.Notpreservedovergeologictime:targetingextantorrecentlydeadlife.

ModelofDNAHydrolysis


260K(-13.3˚C)

240K(-33.3˚C)

220K(-53.3˚C)

400kyatoseveralMya

EuropaOcean

SETG Industry Collaboration


• Research & point of care diagnostics development for NIH, NASA, MIT, Gates Foundation, Biodefense, and direct device sales

• Full partner in SETG MATISSE effort focused on nucleic acid extraction• Developed fully-automated extraction solution (TRL4)• Developed pre-TRL5 automated cartridge• Device heritage: lab, field, NEEMO, ISS (WetLab-2)

SimplePrep™X8OmniLyse®/PureLyse® SimplePrep™X1(TRL4)

RobertDoeblerandMarkBrown@ClaremontBiosolutions,LLC

Pre-TRL5Cartridge

NEEMO ISS(WetLab-2)Field(Iceland)

SETG Industry Collaboration


IonTorrent(Jonathan Rothberg)

MiniaturizableMassivelyparallelLow-power(chip)1Lwaste/run

Genia Technologies,Inc.

AcquiredbyLifeTechnologies,AcquiredbyThermoFisher

AcquiredbyRoche,unabletomeetMATISSEschedule

Time (milliseconds)

Cur

rent

(p

A)

C

T

Sequencing bySynthesis

A

G Monitor ionic current (I)through nanopore(s)

Estimate DNA basesusing statistical models

I

CT

ATagged

Nucleotides

MiniatureMassivelyparallelLow-power(chip)Insituflowcell~1mlwaste/runAccuracyTBDSchedule

DNA Sequencing: Device Comparison


Readlengthlimitunknown(currently>1Mb)

Credit:LexNederbragt https://goo.gl/KhwMQL (2016)

1000kg>7kW

~100g~1W

Strand Sequencing


StrandSequencingOxfordNanopore

Technologies, Inc.(ONT)

Monitor IonicCurrentThroughNanopore(s)

"Basecall”: infersequences ofbasesfromioniccurrenteventsSquiggletoA,T,C,G,U

MotorproteinfunctionslikearatchettopushDNAthroughporebasebybase

2048nanoporearray

Onelibrarycangenerate5-10billionbases,hundredofGiB rawdata.Dataoutputcanexceedentirecommunicationbudget ofsomemissions (e.g.,EuropaLander).⇒ Needin-situdataprocessing!

Image:OxfordNanoporeTechnologies

Library Preparation


1DLigation RapidTransposase-based

SuitableforanylengthofDNA RequiresDNA>10kblengthDNAdamageornon-standardbasesmayinterfere

Imagecredits:OxfordNanoporeTechnologies

Sequencing Lambda Phage


BobDu

dahttps://goo

.gl/sm5hPT

Carretal.(2017)IEEEAerospace.

E.coli CsgG

DetectTranslocation(Ratchet)Events NeuralNetwork-based

DataProcessing

EstimatedDNA

SequenceAnalysis

Lambdahas48.5kbgenome;isusedascontrolfornanoporesequencing.

15

OxfordNanoporecompileddatafrom1000+labstotrainnetwork.


Current SETG Capabilities


Sequencingon“Mars”(manuscriptinprep.)

DetectionofInosine (I)base(nucleoside)

…CICICICIC…

UseofsyntheticMarsanalogs+B.subtilis sporestodemonstrateautomatedextractiondownto104 spores.

Additionalprogress:• “CarrierSeq”:lowinputsequencingdownto2pg inputDNA,equivalentto5%extractionyieldfrom104 sporesin50mg

sediment(1ppbDNA);Mojarro etal.BMCBioinformatics(2018),doi:10.1186/s12859-018-2124-3• “SequencingNothing”:ConfirmationoflittletonofalsepositiverateintheabsenceofDNA(Pontefractetal.,submitted)• ValidationofnanoporesequencingduringparabolicflightunderMars,lunar(Europa),and“0-g”conditions.• SamplingandsequencingofmultipleterrestrialanalogsofMars(SpottedLake,VolcánCopahue,HaughtonImpactStructure)

Nanopore Detection of Inosine


Nucleobasesinmeteorites

• Guanine(G)isastandardnucleobase.• Hypoxanthine isnextmostabundant.• Hypoxanthine+ribose=Inosine

+ =

Ourapproach:• SyntheticDNApolymermadeof

CICICICICIC… poly(dI-dC)• KnowledgethatCsgG nanopore

currentlargelyreflects~3bases.

Callahanetal.(2011)

CCBY-SA3.0

https://goo

.gl/S

GBkXz

17

Meteorite amino acid abundance is likely linked to UV stability (presumed synthesis in solar nebula).

Nanopore Detection of Inosine


24 events in 23.8 s (1.01 events/s)

Lambda:many3-mers

Poly(dI-dC):3-mers:CICorICI

Carretal.IEEEAerospace(2017)

18

ONT-SETG Collaboration

• MinION Early Access Program– Participated briefly, withdrew to work with Genia, returned in

2016– Provided data for early neural network basecaller

• Standard members of Nanopore Community (1000+ labs)– No exchange of $ aside from standard ONT retail practices

• Accommodations to facilitate unique use cases: offline software (field, ISS use), custom part numbers (NASA/ISS), beta testing opportunities

• Contribute to technology transmission through open source code, methods (CarrierSeq), quantification and sharing of process yields (yield of extraction, library preparation/sequencing), use of standardized samples(Mars analogs + B. subtilis spores)

• Future opportunities to leverage ONT-internally developed tools useful for embedded use (command line software) or enhanced function (alternative motor proteins)


MinION (April,2014)

ONT-NASA Collaboration

• NASA: strong track record with well established technologies (e.g., mass spectrometers, optical instruments), in-house and trusted external partners

• Less in-house expertise for emerging technologies: sample preparation, nucleic acid sequencing, other emerging technologies for life detection

• Approach for Biomolecule Sequencer (prior), Genes in Space-3 (current), Biomolecule Extraction and Sequencing Technologies:– ONT most development; joint risk reduction for execution; NASA flight– No exchange of $ aside from standard ONT retail practices– Benefits: proof of concept (NASA), high profile science (NASA, ONT)

• Risk: not owning the technology; industry shifts• Recommendations:

– expand range of acceptable kinds of partnerships for scientific instruments– leverage industry expertise and funding– provide industry development $/incentives to reach specific goals– Consider competition where appropriate to reduce risk

• Examples:– ISS resupply & commercial crew– SpaceX Falcon Heavy vs. SLS– Providing sequencing services on Mars vs. Part of instrument team


PerspectivesfromAaronBurton,PIBiomoleculeSequencer

KateRubins onISS

SpaceX CrewDragon BoeingStarliner

Artistconceptions

AaronBurton

Strand Sequencing: Beyond DNA


ChangeMotorProtein:Enabledifferentsimilarlysizedpolymer(RNAorotherwise)

Testnon-standardbases:Demonstrateexistingmotorproteins/poreswork(manydo)

ChangePore: Enablealternativepolymersizeortype(polypeptide)

ChangeMembrane: ONTplans5-10nmsolidstateporeformeasurementapplications

iImprovealgorithm(ongoing):• inferencefromrawcurrent• trainonalternativepolymers

(eveninsitu)• estimatenotonlysequence

buttaxonomy

Improvehardware:enableacceleratedefficientdataprocessinganywhere

EnhanceDNAdeliverytopore:Canimprove>103(currently10-30basessequencedperMbinput)


Flight Readiness Challenges

• Technology readiness level (TRL) advancement: work around commercial device limitations

• Biological reagents: – Complexity, storage, stability, planetary

protection (bake-out survival)– Radiation protection (Europa)– Solid state sensors would largely eliminate

reagents & library preparation, simplify• Require industry partner buy in for flight

instrument


MinIONReagents

Solid State Sensor: Electron Tunneling


QuantumElectronTunneling(QET)DetectionandSequencingUsingSolidStateNanogaps. (A)Nanogap concept. (B)Functionalsystem.(C)QETcurrentsignalshowingregionscorrespondingtooligo sequences. (D)Detection ofTGAGTAGTAGTGTATA. (E)Currenthistogramsassociated withspecificDNAbases.AdaptedfromQuantumBiosystems; technology contributions fromMasateru Taniguchi, OsakaUniversity.

ONTisalsoworkingonsolidstatesequencing (fundingacademic labs).

Solid State Senor: Electron Tunneling


Masateru TaniguchiLab,OsakaUniversity,©NatureNanotechnologyhttps://goo.gl/bK812P

Nanogap previouslyusedtodetectDNA,RNA,nowappliedtosingleaminoacids:

Solid State Senor: Electron Tunneling


Masateru TaniguchiLab,OsakaUniversity,©NatureNanotechnologyhttps://goo.gl/bK812P

Nanogap previouslyusedtodetectDNA,RNA,nowappliedtosingleaminoacids:

0.55nmgap• Nanogap canbe

adaptedtomultiplepolymerandmoleculetypes.

• Machinelearning canhelpdistinguishinformationcontentbeyondconductanceandtime.

• Noisesuppressionandcontrolovermolecularconformation critical.

Commercial Case for Sequencing Non-standard bases & Polymers

• Press/Public Relations– e.g., in association with life detection mission– example: sequencing on ISS

• Strategic Investment/Intellectual Property– Non-standard bases and polymers used in

diagnostics (patentability)– Clinical relevance (methylation)– Synthetic biology/defense (kill switch, orthogonal life)– Establish broad barriers to entry (patents)

• Intellectual Reputation– Attract/retain talent; corporate pride

• Immortality– Potential to be a part of detecting life beyond Earth


Recommendations

• Targeting nucleic acids and informational polymers is a critical part of a comprehensive life detection approach.

• Mars: high chance of ancestrally related life; elsewhere, potential for common physicochemical origins; need to detect forward contamination

• Life detection missions: need improved autonomy to acquire the best samples.• Current life detection approaches for nanopore sequencing and nanogap single

molecule detection require machine learning (ML) / neural network approaches.• In situ data reduction is essential, and must be efficient: opportunity for multi-

purpose ML data processors for space applications.• Space agencies should leverage industrial R&D in “AI” and molecular sensing,

including international collaboration – many relevant companies are not in the US.• NASA already using SBIR/STTR mechanisms: improve matching of industry

innovators to opportunities; offer funding without equity stake but with commitment of technology to Astrobiology/NASA applications?

• Improve robustness Earth-based autonomous systems and usability of environmental and clinical diagnostics.


Questions?


?

Mars Regolith

NASA/JPL-Caltech/TedStryk/SpaceScienceInstitute

Selected Publications (2017-2018)• Full list: http://setg.mit.edu/publications/• Mojarro A, Hachey J, Ruvkun G, Zuber MT, Carr CE. CarrierSeq: a sequence analysis workflow for low-input nanopore

sequencing. BMC Bioinformatics, 2018, 19:108, doi: 10.1186/s12859-018-2124-3• Pontefract A, Zhu TF, Walker VK, Rowedder H, Lui C, Zuber MT, Ruvkun G, Carr CE. Microbial Diversity in a Hypersaline Sulfate

Lake: An Analog of Ancient Mars. Frontiers in Microbiology, 26 Sept 2017 doi: 10.3389/fmicb.2017.01819• Mojarro A, Ruvkun G, Zuber MT, Carr CE. Nucleic acid extraction from synthetic Mars analog soils for in situ life detection.

Astrobiology 2017 Jul 13. doi: 10.1089/ast.2016.1535. • 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 doi:10.1109/AERO.2017.7943896 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


single molecule sensing and sequencing for life detection

Documents