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Nanopore sequencing at Mars, Europa, and microgravity conditions

Nanopore sequencing at Mars, Europa, and microgravity conditions www.nature.com/npjmgrav ARTICLE OPEN Nanopore sequencing at Mars, Europa, and microgravity conditions 1,2,4✉ 1 1 3 2 1 Christopher E. Carr , Noelle C. Bryan , Kendall N. Saboda , Srinivasa A. Bhattaru , Gary Ruvkun and Maria T. Zuber Nanopore sequencing, as represented by Oxford Nanopore Technologies’ MinION, is a promising technology for in situ life detection and for microbial monitoring including in support of human space exploration, due to its small size, low mass (~100 g) and low power (~1 W). Now ubiquitous on Earth and previously demonstrated on the International Space Station (ISS), nanopore sequencing involves translocation of DNA through a biological nanopore on timescales of milliseconds per base. Nanopore sequencing is now being done in both controlled lab settings as well as in diverse environments that include ground, air, and space vehicles. Future space missions may also utilize nanopore sequencing in reduced gravity environments, such as in the search for life on Mars (Earth-relative gravito-inertial acceleration (GIA) g = 0.378), or at icy moons such as Europa (g = 0.134) or Enceladus (g = 0.012). We confirm the ability to sequence at Mars as well as near Europa or Lunar (g = 0.166) and lower g levels, demonstrate the functionality of updated chemistry and sequencing protocols under parabolic flight, and reveal consistent performance across g level, during dynamic accelerations, and despite vibrations with significant power at translocation-relevant frequencies. Our work strengthens the use case for nanopore sequencing in dynamic environments on Earth and in space, including as part of the search for nucleic-acid based life beyond Earth. npj Microgravity (2020) 6:24 ; https://doi.org/10.1038/s41526-020-00113-9 INTRODUCTION “transition”, “parabola”, “hypergravity”, and “other” (typically, gentle climb, descent, straight and level flight, or standard rate Life as we know it uses nucleic acids as the basis for heredity and turns) on the basis of accelerometer measurements . Sequencing evolution. Life beyond Earth might utilize identical or similar was also performed on the ground prior to the flight as a control. informational polymers due to the widespread synthesis of common building blocks, common physicochemical scenarios for life’s origin(s), or common ancestry via meteoritic exchange, Sequencing most plausible for Earth and Mars. Beyond the search for life, Sequencing of control lambda deoxyribonucleic acid (DNA) was sequencing is of high relevance for supporting human health on performed for a total of 38 min on the ground and 103 min during Earth and in space, from detecting infectious diseases, to flight, on the same flow cell, resulting in 5293 and 18,233 reads for monitoring of biologically-based life support systems. ground (Supplementary Fig. 1) and flight (Fig. 1c; Supplementary Nanopore sequencing , as commercialized by Oxford Nanopore Fig. 1) respectively, of which 5257 and 18,188 were basecalled Technologies, is a promising approach that is now used (Supplementary Tables 1, 2). Of the flight reads, 14,431 fell wholly ubiquitously in the lab and in the field. McIntyre et al. reported within a phase of flight, including parabola (404), hypergravity a single mapped read obtained via nanopore sequencing during (1996), transition (7), and other (12,024). Sequencing reads were parabolic flight, obtained across multiple parabolas . Vibration of obtained during all parabolas, including under Mars, lunar/Europa, flow cells revealed that 70% of pores should survive launch, and zero-g conditions (Fig. 2). The g levels achieved during each consistent with later successful nanopore sequencing on the parabola were previously reported . For the purposes of statistical International Space Station (ISS) . However, we are not aware of analysis, mux reads (Fig. 1c, black horizontal lines) were excluded any nanopore experiments that attempted to quantify the impact to avoid any sequencer start-up effects. of vibration while sequencing. Here we test the impacts of: (1) altered g level, (2) vibration, and (3) updated chemistry/flow cells. Vibration Zero-phase filtering effectively removed frequencies at or below 10 Hz (Supplementary Figs 2–6). Filtered root-mean-square (RMS) RESULTS vibration varied throughout the flight and showed clear deviations Parabolic flight associated with parabolas (Figs 1c, 2a; Supplementary Fig. 2), indicating a smoother environment during freefall. Remaining Flight operations were conducted on November 17, 2017 onboard aircraft-associated vibrations were largely in the 10 Hz to 1 kHz a Boeing 727-200F aircraft (G-Force One®, Zero Gravity Corpora- band with peaks at 116–128, 250–270, 495–496, 580–680, and tion). Four sets of parabolas were performed with 5, 6, 4, and 5 876 Hz (Supplementary Fig. 6). During zero-g parabolas, the parabolas respectively (Fig. 1a). The first set targeted, in order, Mars g, Mars g, Lunar g,0 g, and 0 g (Fig. 1b). All other parabolas magnitude of the residual g level and vibrations were comparable targeted 0 g. The flight profile was segmented into periods of (Fig. 2a). 1 2 Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Molecular Biology, Massachusetts 3 4 General Hospital, Boston, MA, USA. Department of Aeronautics and Astronautics, Massachusetts Institute of Technology, Cambridge, MA, USA. Present address: Georgia Institute of Technology, ESM Building, Room G10, 620 Cherry St NW, Atlanta, GA 30332, USA. email: cecarr@gatech.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; C.E. Carr et al. By aligning ionic current signals to bases, tombo allowed us to measure the translocation time associated with each base (Supplementary Fig. 9), the time required for the motor protein, acting as a ratchet, to move the DNA strand one base into the nanopore. Translocation here refers to motion of the motor protein relative to the DNA strand, and not the total time to get through the nanopore, which requires many translocation steps. The inverse of translocation time is a direct measurement of sequencing rate for a given nanopore (bases/s). Translocation times were similar, but statistically longer, during flight as compared to ground. Despite the nearly sixfold (5.89) average higher RMS vibration during flight compared to ground (Fig. 3a), the probability densities for translocation time are strikingly similar (Fig. 3b; Supplementary Fig. 9). However, base translocation times were significantly different (Kolmogorov–Smirnov, two-tailed, p = 0, test statistic 0.0306), with a slight shift toward longer translocation times during flight. Notably, the median base translocation times were identical (seven samples or 1.8 ms) and the means only differed by 0.125 ms (2.2786 ms, ground; 2.4035 ms, flight). Thus, translocation times were robust to large variations in vibration. Ionic current noise is the variation in the flow of ions passing through the nanopore, measured here at the per-base level as a normalized signal standard deviation determined by tombo through optimal alignment of measured ionic current to a Fig. 1 Single molecule sequencing during parabolic flight. a genomic sequence . A stepwise linear regression was performed Phases of flight timeline (black: other/1 g; red: hypergravity; to determine if time, RMS vibration, or their combined effects magenta: transition; blue: parabola). b Phases of flight for first set were significant predictors of ionic current noise during ground of parabolas. c Vibration (blue line, left axis) and sequencing reads (Supplementary Fig. 10; Supplementary Table 7) and flight measured during flight; each read is represented by a horizontal line (Supplementary Fig. 10; Supplementary Table 8) operations. Flight (mux = black, run = red) at its representative read quality score, q . analysis included the additional variable g level. For ground operations, the impact of time alone was not significant. However, both vibration (p = 0.0018) and the interac- Integrated read-level analysis tion effect of time and vibration (p = 0.041) were significant Stepwise linear regression was used to determine whether time predictors of ionic current noise (Supplementary Table 7). and RMS vibration could predict median sequence quality 5,6 However, the explanatory power of the regression was low (adj. (Supplementary Fig. 1), the Phred quality score associated with R = 0.009). Conversely, time was the only significant predictor of the average per-base error probability of a given read (see the effect on ionic current noise during flight. Neither RMS Materials and Methods). Unlike ground operations, where time vibration, g level, nor any of their respective combined effects had was the only significant predictor of sequence read quality (p = 0), significant impacts on ionic current noise (Fig. 4; Supplementary time, g level, and their combined effects were predicted to be Table 8). −4 significant indicators during flight (all p <10 ; Supplementary Because time was a significant indicator of ionic current noise Tables 3, 4). However, in both cases, the variance explained was during flight, it was necessary to assess whether the effect could small (adj. R = 0.060 and 0.275, respectively, for ground and be attributed to a specific phase of flight (Supplementary Table 9). flight). Tukey’s HSD post hoc test demonstrated that out of all six possible In order to elucidate the role of g level on read quality, those pairwise comparisons, only one, parabola vs. transition, was not −3 reads falling wholly within an individual phase of flight were significant (two-sided p = 0.345; other p <10 ). Ionic current was examined using a one-way ANOVA, with Tukey’s Honest significantly lower in hypergravity, parabola, and transition phases Significant Difference (HSD) post hoc analyses (Supplementary as compared to other. Ionic currents during hypergravity phases Table 5). Sequence quality was significantly different during each were, on average, lower than all other phases (Supplementary phase of flight, with the lowest read quality during parabolas Table 9; Supplementary Fig. 11). Thus, while the impact of phase (q = 8.3) and the highest quality (q = 8.7) during hypergravity of flight on read quality showed a trend toward higher read p p (Supplementary Fig. 7). quality with higher g level (Supplementary Fig. 7), no such pattern was observed with ionic current (Supplementary Fig. 11). Integrated base-level analysis Tombo was used to associate raw ionic current signals with DISCUSSION specific genomic bases, and the number of reads aligning was The Mars 2020 rover, currently enroute to Mars, is expected to 5,6 similar to the number of reads with Phred quality scores >6.5. touch down in Jezero Crater in February, 2021. While this mission The percentage of bases that aligned to the lambda genome via will not attempt to detect extant life, it represents a new era in the tombo was 87.8% and 89.7% for ground and flight, respectively search for life beyond Earth. Ambiguous or positive results in the (Supplementary Table 1). Average coverage for tombo-aligned search for ancient life could usher in a new era of life detection bases was adequate to sequence the lambda genome many times efforts, including instrumentation aimed at measuring the over during each parabola (Fig. 2d) and the coverage was largely presence of nucleic acids, one of the “smoking gun” pieces of explained by parabola duration (adj. R = 0.807; Supplementary evidence for life beyond Earth . In preparation for future life Table 6). Ionic current levels associated with unique subsequences detection missions targeting DNA, we explored the capabilities of (k-mers) were similar between ground and flight conditions nanopore sequencing, and present results demonstrating its (Supplementary Fig. 8). successful performance while experiencing aircraft vibrations npj Microgravity (2020) 24 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; C.E. Carr et al. Fig. 2 Sequencing in reduced gravity. a g level achieved (black line) and RMS vibration (1 s bins, blue line) and associated sequencing reads acquired during first set of parabolas. Each read is represented by a horizontal line (gray: partially or completely in transition period; red: completely in non-transition period) at its representative read quality score, q (right axis). Vertical gray bands demarcate transitions between phases of flight. b Top scoring BLAST results for highest quality “Mars” read, indicated via arrow in a, length 6402. c Start of best match sequence alignment, to J02459.1 Enterobacteria phage lambda, complete genome, length 48502 (range 20562–27113, score 8907 bits(9877), expect 0.0, identities 6108/6651 (92%), gaps 395/6651 (5%), strand Plus/Minus). d Average genomic coverage of lambda for all parabolas based on tombo-aligned bases. Fig. 3 Translocation time is weakly or not affected by vibration. a RMS vibration distributions for ground and flight. b Nanopore translocation time as measured by alignment of ionic current to the genomic reference: distribution for <10 ms. Ground (blue), flight (light brown), both (dark brown). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 24 C.E. Carr et al. smallest mean values of ionic current noise were also observed during hypergravity (0.282; Supplementary Fig. 11). Although statistically larger, the difference between largest mean value for ionic current noise (phase other) was miniscule (0.003). Nanopore sequencing is compatible with many life detection missions from the perspective of mass (~100 g), size, and power (<2 W). Recent work also suggests that MinION electronics and flow cell components would survive radiation doses consistent with life detection missions to Mars, Venus, and Enceladus, although not Europa, without additional shielding . Our work shows that sequencing on all these worlds, including Europa, could be feasible from a g level perspective. In addition, the robustness to vibration suggests that operation concurrent with other mission activities, such as drilling or operation of other instrument payloads, could occur without any substantial negative impacts. In addition, our work highlights the potential for nanopore sequencing on Earth and beyond in mobile and dynamic environments such as on passenger aircraft, drones, wheeled vehicles, ships, buoys, underwater vehicles, or other Fig. 4 RMS vibration and median ionic current noise during flight. platforms. The single 1 s period with median ionic current noise >0.5 has a median absolute deviation (MAD) of >15 and is therefore an outlier (typically defined as MAD >3). METHODS and under altered g levels, including those that would be Acceleration measurement and flight profile segmentation encountered on the surface of Mars, the Moon, and/or Jupiter’s The flight profile was segmented as described in Carr et al. from moon Europa. Due to the limitations of parabolic flight, our zero-g acceleration data collected using a metal-body Slam Stick X™ (Mide Technology Corp.). The accelerometer was mounted next to the MinION to conditions involved mean acceleration around 4× higher (0.041 ± a common baseplate, using double sided sticky tape (3M 950) to provide a 0.005 g) than at the surface of Saturn’s moon Enceladus (0.011 g). near-unity vibration frequency response. Vibrations were measured with Several factors may have influenced the overall quality of our the internal triaxial piezoelectric accelerometer (TE Connectivity Ltd., nanopore sequencing data. The DNA sequencing library used in 832M1) at a frequency of 5 kHz. this experiment was stored for 72 h prior to loading onto the flow cell. As such, the sample was potentially subjected to degradation, Sequencing which could impact read quality, and may have resulted in the loss Sequencing libraries were prepared using DNA derived from Enterobacteria of ligated adaptors. Such conditions would negatively impact the phage lambda (NEB N3011S), fragmented using a g-TUBE™(Covaris® proper loading of DNA into the individual nanopore. In addition, 520079) with the 6 kb protocol. Next, the libraries were prepared using there is an expected degradation of the flow cell over time during the 1D ligation method (SQK-LSK108) using a “one-pot” barcoding sequencing, which could explain some of the time-related trends, protocol and stored at 4 °C for ~72 h prior to the flight. At the time of independent of any effects of vibration or acceleration. Despite storage, the total library DNA was estimated to be 440 ng at 31.4 ng/μlas sequencing for a limited time at any given g level during parabolic assessed by fluorometry (ThermoFisher Qubit® 3.0 Fluorometer with flight, the operation of the MinION for sustained periods on the Qubit™ dsDNA HS Assay Kit, Q32854). ISS gives us confidence that extended periods of reduced g level A flow cell (FLO-MIN106 R9) was loaded on the ground and sequencing does not negatively impact nanopore sequencing. In addition, it performed using an offline version of MinKNOW 1.7.14 in the flight provides confidence in nanopore sequencing as a viable life hardware configuration while the aircraft was on the ground. After 38 min, sequencing was stopped. In flight, sequencing was reinitiated around detection technology in very low but nonzero g environments, 12 min prior to parabolic flight maneuvers, and continued for a total of such as Enceladus. 103 min before termination. After the flight, basecalling was performed Because zero-phase filtering of vibration data effectively with ONT Albacore version 2.3.1 with quality filtering disabled. removed frequencies at or below 10 Hz (Supplementary Figs 5, 6), filtered vibration measurements did not reflect frequencies Sequence data processing where sensor data would be inaccurate due to the non-unity frequency response of the sensor near DC (0 Hz). In addition, this To quantify adaptor sequences, fastq output was trimmed using Porechop (https://github.com/rrwick/Porechop docker container quay.io/biocontainers/ filtering ensured that we could assess the independent effects of porechop:0.2.3_seqan2.1.1-py35_2). Original untrimmed fast5 reads were g level and vibration. aligned to the reference genome (NEB Lambda, equivalent to NCBI The peaks in the vibration spectrum occurs at frequencies NC_001416.1 with mutations 37589 C→T, 45352 G→A, 37742 C→T, and relevant to nanopore sequencing. Despite this, vibration did have 43082 G→A) using tombo (docker container quay.io/biocontainers/ont- any significant impacts on sequence quality nor on ionic current tombo:1.5-py27r351h24bf2e0_0) with the—include-event-stdev option. noise, except during ground-based sequencing, where the explanatory power of vibration was negligible (<1%; Supplemen- Sequencing and acceleration data integration tary Table 7). A custom script was used to parse tombo-processed fast5 files to Random vibration at translocation-relative frequencies could characterize each read and each tombo-aligned genomic base within each exert a minor interfering effect on translocation, although any read (Supplementary Data). A representative read quality score was impact in changes in vibration during flight did not translate into calculated as q ¼10log ðpÞ, where p is the mean of the per-base error p 10 any consistent or large changes in ionic current noise due to the −q/10 5,6 probability p = 10 , where q is the per-base Phred quality score small (0.125 ms) mean difference in translocation times despite estimated via basecalling. Read timings were adjusted by offsets to align nearly a sixfold change in RMS vibration. genomic and accelerometer data (Supplementary Table 2). Each read and Higher g levels tended to be associated with higher read base was assigned one of the following states (parabola, transition, quality, although the effect size is small (Δq = 0.4, hypergravity − hypergravity, other) on the basis of the periods.txt file produced by prior parabola; Supplementary Table 5, an upper bound of ~0.2/g). The analysis and available online at https://osf.io/nk2w4/. npj Microgravity (2020) 24 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA C.E. Carr et al. Vibration data processing Preprint A vibration equivalent to g level (Earth-relative gravity) was computed A previous version of this manuscript was published as a preprint . qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 as g ¼ g þ g þ g to provide a measure of vibration that is x y z Reporting summary independent of the Slam Stick X™ orientation. The vibration power spectral density (PSD) for g was computed using Further information on research design is available in the Nature Research Welch’s method (MATLAB pwelch() function) with default parameters Reporting Summary linked to this article. (Supplementary Fig. 4). Filtering was then performed for two reasons: (1) to eliminate vibration data where the frequency response of the piezoelectric accelerometer is not unity, and (2) to analyze vibration at frequencies DATA AVAILABILITY related to timescales at which base translocation occurs during nanopore Raw and calibrated data are available via the Open Science Framework at: sequencing, which are overwhelmingly <10 ms (Fig. 3b, Supplementary https://osf.io/n6krq/ and https://osf.io/nk2w4/. Fig. 9). The g level equivalent vibration g was filtered with a high pass infinite impulse response filter (Supplementary Fig. 5) that was generated with MATLAB’s designfilt() function (stopband 5 Hz @ 60 dB attenuation, CODE AVAILABILITY passband 10 Hz with unity ripple, sample rate 5 kHz). Filtering was The MATLAB scripts implementing our analysis are available at: performed using the MATLAB filtfilt() function, which uses forward and https://github.com/CarrCE/zerogseq. reverse filtering to achieve zero-phase delay. The PSD was computed as before for the resulting filtered g level equivalent vibration g (Supple- Received: 10 January 2020; Accepted: 11 August 2020; mentary Fig. 6). RMS vibration was computed in 1 s bins from g using the MATLAB rms() fuction. An overview of vibration is shown in Supplementary Fig. 2 for flight and Supplementary Fig. 3 for ground. Sequencing read quality regression analysis REFERENCES Sequencing read times were adjusted by an offset to place sequencing 1. Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford nanopore MinION: reads into the accelerometer elapsed time (Supplementary Table 2). A time delivery of nanopore sequencing to the genomics community. Genome Biol. 17, series of median read quality was estimated in 1 s bins by computing the 239 (2016). median of q for all reads covering the bin. Stepwise linear regression, via 2. McIntyre, A. B. R. et al. Nanopore sequencing in microgravity. npj Microgravity 2, the MATLAB stepwiselm() function, was used to evaluate the impact of time, 16035 (2016). RMS vibration, and g level (flight only) on sequence quality, as measured by 3. Castro-Wallace, S. L. et al. Nanopore DNA sequencing and genome assembly on median q (Supplementary Tables 3, 4). For flight, the regression time was the International Space Station. Sci. Rep. 7, 18022 (2017). restricted to a maximum elapsed time of 4000 s to eliminate potential 4. Carr, C. E. et al. Acceleration profiles and processing methods for parabolic flight. confounding effects of the aircraft descent and landing. npj Microgravity 4, 14 (2018). 5. Ewing, B., Hillier, L., Wendl, M. C. & Green, P. Base-calling of automated sequencer Sequencing read quality phase of flight analysis (flight only) traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185 (1998). 6. Ewing, B. & Green, P. Base-calling of automated sequencer traces using phred. II. To assess differences in read quality as a function of phase of flight, we Error probabilities. Genome Res. 8, 186–194 (1998). performed a one-way analysis of variance (ANOVA) via the MATLAB anova1() 7. Stoiber, M. H. et al. De novo identification of DNA modifications enabled by function on the non-mux reads (Supplementary Table 5), excluding reads in genome-guided nanopore signal processing. Preprint at http://biorxiv.org/ transition periods due to their low number (7) and short length. To compare lookup/doi/10.1101/094672 (2016). group means we then used Tukey’sHonestly Significant Difference test 8. Neveu, M., Hays, L. E., Voytek, M. A., New, M. H. & Schulte, M. D. The ladder of life (MATLAB multcompare() function), which accounts for multiple testing and is detection. Astrobiology 18, 1375–1402 (2018). conservative for one-way ANOVA with different sample sizes. 9. Sutton, M. A. et al. Radiation tolerance of Nanopore sequencing technology for life detection on Mars and Europa. Sci. Rep. 9, 5370 (2019). Coverage of genomic-aligned bases 10. Quick, J. One-pot ligation protocol for Oxford Nanopore libraries. Preprint at https://doi.org/10.17504/protocols.io.k9acz2e (2018). Base times were adjusted by an offset to place each tombo-aligned base 11. Carr, C.E. et al. Nanopore sequencing at Mars, Europa, and microgravity condi- into the accelerometer elapsed time (Supplementary Table 2). Coverage tions. Preprint at https://www.biorxiv.org/content/10.1101/2020.01.09.899716v1. was estimated as the sum of tombo-aligned bases within a given phase of flight divided by the lambda genome size (48,502 bases). Stepwise linear regression, via the MATLAB stepwiselm() function, was used to evaluate ACKNOWLEDGEMENTS the relationship between coverage and parabola period (Supplementary Table 6). We thank the MIT Media Lab Space Exploration Initiative for providing the parabolic flight. This work was supported by NASA award NNX15AF85G. N.C.B. was supported by NASA Postdoctoral Fellowship award 80NSSC17K0688. Base ionic current noise regression analysis A time series of ionic current noise was estimated in 1 s bins by computing the median of ionic current (tombo norm_std output) for all bases within a AUTHOR CONTRIBUTIONS bin. Stepwise linear regression, via the MATLAB stepwiselm() function, was C.E.C. designed the experiment, C.E.C., K.S., S.A.B., and N.C.B. built and tested the used to evaluate the impact of time, RMS vibration, and g level (flight only) hardware, N.C.B. and M.T.Z. collected the data, C.E.C. and N.C.B. processed the data. on median ionic current noise (Supplementary Tables 7, 8). For flight, the G.R. advised on the experiment design. C.E.C. and N.C.B. wrote, and all authors edited regression time was restricted as stated above. and approved, the paper. C.E.C. is the guarantor. Base ionic current noise phase of flight analysis (flight only) CONFLICT OF INTEREST To assess differences in ionic current as a function of phase of flight, we performed a one-way ANOVA via the MATLAB anova1() function on the The authors declare no conflict of interest. non-mux tombo-aligned bases (Supplementary Table 9). To compare group means we then used Tukey’s Honestly Significant Difference test as above. ADDITIONAL INFORMATION Supplementary information is available for this paper at https://doi.org/10.1038/ Does flight vs. ground impact translocation time? s41526-020-00113-9. A Kolmogorov–Smirnov test was performed with the MATLAB kstest2() Correspondence and requests for materials should be addressed to C.E.C. function on the base translocation times for ground vs. flight. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 24 C.E. Carr et al. appropriate credit to the original author(s) and the source, provide a link to the Creative Reprints and permission information is available at http://www.nature.com/ Commons license, and indicate if changes were made. The images or other third party reprints material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims article’s Creative Commons license and your intended use is not permitted by statutory in published maps and institutional affiliations. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give © The Author(s) 2020 npj Microgravity (2020) 24 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals

Nanopore sequencing at Mars, Europa, and microgravity conditions

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www.nature.com/npjmgrav ARTICLE OPEN Nanopore sequencing at Mars, Europa, and microgravity conditions 1,2,4✉ 1 1 3 2 1 Christopher E. Carr , Noelle C. Bryan , Kendall N. Saboda , Srinivasa A. Bhattaru , Gary Ruvkun and Maria T. Zuber Nanopore sequencing, as represented by Oxford Nanopore Technologies’ MinION, is a promising technology for in situ life detection and for microbial monitoring including in support of human space exploration, due to its small size, low mass (~100 g) and low power (~1 W). Now ubiquitous on Earth and previously demonstrated on the International Space Station (ISS), nanopore sequencing involves translocation of DNA through a biological nanopore on timescales of milliseconds per base. Nanopore sequencing is now being done in both controlled lab settings as well as in diverse environments that include ground, air, and space vehicles. Future space missions may also utilize nanopore sequencing in reduced gravity environments, such as in the search for life on Mars (Earth-relative gravito-inertial acceleration (GIA) g = 0.378), or at icy moons such as Europa (g = 0.134) or Enceladus (g = 0.012). We confirm the ability to sequence at Mars as well as near Europa or Lunar (g = 0.166) and lower g levels, demonstrate the functionality of updated chemistry and sequencing protocols under parabolic flight, and reveal consistent performance across g level, during dynamic accelerations, and despite vibrations with significant power at translocation-relevant frequencies. Our work strengthens the use case for nanopore sequencing in dynamic environments on Earth and in space, including as part of the search for nucleic-acid based life beyond Earth. npj Microgravity (2020) 6:24 ; https://doi.org/10.1038/s41526-020-00113-9 INTRODUCTION “transition”, “parabola”, “hypergravity”, and “other” (typically, gentle climb, descent, straight and level flight, or standard rate Life as we know it uses nucleic acids as the basis for heredity and turns) on the basis of accelerometer measurements . Sequencing evolution. Life beyond Earth might utilize identical or similar was also performed on the ground prior to the flight as a control. informational polymers due to the widespread synthesis of common building blocks, common physicochemical scenarios for life’s origin(s), or common ancestry via meteoritic exchange, Sequencing most plausible for Earth and Mars. Beyond the search for life, Sequencing of control lambda deoxyribonucleic acid (DNA) was sequencing is of high relevance for supporting human health on performed for a total of 38 min on the ground and 103 min during Earth and in space, from detecting infectious diseases, to flight, on the same flow cell, resulting in 5293 and 18,233 reads for monitoring of biologically-based life support systems. ground (Supplementary Fig. 1) and flight (Fig. 1c; Supplementary Nanopore sequencing , as commercialized by Oxford Nanopore Fig. 1) respectively, of which 5257 and 18,188 were basecalled Technologies, is a promising approach that is now used (Supplementary Tables 1, 2). Of the flight reads, 14,431 fell wholly ubiquitously in the lab and in the field. McIntyre et al. reported within a phase of flight, including parabola (404), hypergravity a single mapped read obtained via nanopore sequencing during (1996), transition (7), and other (12,024). Sequencing reads were parabolic flight, obtained across multiple parabolas . Vibration of obtained during all parabolas, including under Mars, lunar/Europa, flow cells revealed that 70% of pores should survive launch, and zero-g conditions (Fig. 2). The g levels achieved during each consistent with later successful nanopore sequencing on the parabola were previously reported . For the purposes of statistical International Space Station (ISS) . However, we are not aware of analysis, mux reads (Fig. 1c, black horizontal lines) were excluded any nanopore experiments that attempted to quantify the impact to avoid any sequencer start-up effects. of vibration while sequencing. Here we test the impacts of: (1) altered g level, (2) vibration, and (3) updated chemistry/flow cells. Vibration Zero-phase filtering effectively removed frequencies at or below 10 Hz (Supplementary Figs 2–6). Filtered root-mean-square (RMS) RESULTS vibration varied throughout the flight and showed clear deviations Parabolic flight associated with parabolas (Figs 1c, 2a; Supplementary Fig. 2), indicating a smoother environment during freefall. Remaining Flight operations were conducted on November 17, 2017 onboard aircraft-associated vibrations were largely in the 10 Hz to 1 kHz a Boeing 727-200F aircraft (G-Force One®, Zero Gravity Corpora- band with peaks at 116–128, 250–270, 495–496, 580–680, and tion). Four sets of parabolas were performed with 5, 6, 4, and 5 876 Hz (Supplementary Fig. 6). During zero-g parabolas, the parabolas respectively (Fig. 1a). The first set targeted, in order, Mars g, Mars g, Lunar g,0 g, and 0 g (Fig. 1b). All other parabolas magnitude of the residual g level and vibrations were comparable targeted 0 g. The flight profile was segmented into periods of (Fig. 2a). 1 2 Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA. Department of Molecular Biology, Massachusetts 3 4 General Hospital, Boston, MA, USA. Department of Aeronautics and Astronautics, Massachusetts Institute of Technology, Cambridge, MA, USA. Present address: Georgia Institute of Technology, ESM Building, Room G10, 620 Cherry St NW, Atlanta, GA 30332, USA. email: cecarr@gatech.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; C.E. Carr et al. By aligning ionic current signals to bases, tombo allowed us to measure the translocation time associated with each base (Supplementary Fig. 9), the time required for the motor protein, acting as a ratchet, to move the DNA strand one base into the nanopore. Translocation here refers to motion of the motor protein relative to the DNA strand, and not the total time to get through the nanopore, which requires many translocation steps. The inverse of translocation time is a direct measurement of sequencing rate for a given nanopore (bases/s). Translocation times were similar, but statistically longer, during flight as compared to ground. Despite the nearly sixfold (5.89) average higher RMS vibration during flight compared to ground (Fig. 3a), the probability densities for translocation time are strikingly similar (Fig. 3b; Supplementary Fig. 9). However, base translocation times were significantly different (Kolmogorov–Smirnov, two-tailed, p = 0, test statistic 0.0306), with a slight shift toward longer translocation times during flight. Notably, the median base translocation times were identical (seven samples or 1.8 ms) and the means only differed by 0.125 ms (2.2786 ms, ground; 2.4035 ms, flight). Thus, translocation times were robust to large variations in vibration. Ionic current noise is the variation in the flow of ions passing through the nanopore, measured here at the per-base level as a normalized signal standard deviation determined by tombo through optimal alignment of measured ionic current to a Fig. 1 Single molecule sequencing during parabolic flight. a genomic sequence . A stepwise linear regression was performed Phases of flight timeline (black: other/1 g; red: hypergravity; to determine if time, RMS vibration, or their combined effects magenta: transition; blue: parabola). b Phases of flight for first set were significant predictors of ionic current noise during ground of parabolas. c Vibration (blue line, left axis) and sequencing reads (Supplementary Fig. 10; Supplementary Table 7) and flight measured during flight; each read is represented by a horizontal line (Supplementary Fig. 10; Supplementary Table 8) operations. Flight (mux = black, run = red) at its representative read quality score, q . analysis included the additional variable g level. For ground operations, the impact of time alone was not significant. However, both vibration (p = 0.0018) and the interac- Integrated read-level analysis tion effect of time and vibration (p = 0.041) were significant Stepwise linear regression was used to determine whether time predictors of ionic current noise (Supplementary Table 7). and RMS vibration could predict median sequence quality 5,6 However, the explanatory power of the regression was low (adj. (Supplementary Fig. 1), the Phred quality score associated with R = 0.009). Conversely, time was the only significant predictor of the average per-base error probability of a given read (see the effect on ionic current noise during flight. Neither RMS Materials and Methods). Unlike ground operations, where time vibration, g level, nor any of their respective combined effects had was the only significant predictor of sequence read quality (p = 0), significant impacts on ionic current noise (Fig. 4; Supplementary time, g level, and their combined effects were predicted to be Table 8). −4 significant indicators during flight (all p <10 ; Supplementary Because time was a significant indicator of ionic current noise Tables 3, 4). However, in both cases, the variance explained was during flight, it was necessary to assess whether the effect could small (adj. R = 0.060 and 0.275, respectively, for ground and be attributed to a specific phase of flight (Supplementary Table 9). flight). Tukey’s HSD post hoc test demonstrated that out of all six possible In order to elucidate the role of g level on read quality, those pairwise comparisons, only one, parabola vs. transition, was not −3 reads falling wholly within an individual phase of flight were significant (two-sided p = 0.345; other p <10 ). Ionic current was examined using a one-way ANOVA, with Tukey’s Honest significantly lower in hypergravity, parabola, and transition phases Significant Difference (HSD) post hoc analyses (Supplementary as compared to other. Ionic currents during hypergravity phases Table 5). Sequence quality was significantly different during each were, on average, lower than all other phases (Supplementary phase of flight, with the lowest read quality during parabolas Table 9; Supplementary Fig. 11). Thus, while the impact of phase (q = 8.3) and the highest quality (q = 8.7) during hypergravity of flight on read quality showed a trend toward higher read p p (Supplementary Fig. 7). quality with higher g level (Supplementary Fig. 7), no such pattern was observed with ionic current (Supplementary Fig. 11). Integrated base-level analysis Tombo was used to associate raw ionic current signals with DISCUSSION specific genomic bases, and the number of reads aligning was The Mars 2020 rover, currently enroute to Mars, is expected to 5,6 similar to the number of reads with Phred quality scores >6.5. touch down in Jezero Crater in February, 2021. While this mission The percentage of bases that aligned to the lambda genome via will not attempt to detect extant life, it represents a new era in the tombo was 87.8% and 89.7% for ground and flight, respectively search for life beyond Earth. Ambiguous or positive results in the (Supplementary Table 1). Average coverage for tombo-aligned search for ancient life could usher in a new era of life detection bases was adequate to sequence the lambda genome many times efforts, including instrumentation aimed at measuring the over during each parabola (Fig. 2d) and the coverage was largely presence of nucleic acids, one of the “smoking gun” pieces of explained by parabola duration (adj. R = 0.807; Supplementary evidence for life beyond Earth . In preparation for future life Table 6). Ionic current levels associated with unique subsequences detection missions targeting DNA, we explored the capabilities of (k-mers) were similar between ground and flight conditions nanopore sequencing, and present results demonstrating its (Supplementary Fig. 8). successful performance while experiencing aircraft vibrations npj Microgravity (2020) 24 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; C.E. Carr et al. Fig. 2 Sequencing in reduced gravity. a g level achieved (black line) and RMS vibration (1 s bins, blue line) and associated sequencing reads acquired during first set of parabolas. Each read is represented by a horizontal line (gray: partially or completely in transition period; red: completely in non-transition period) at its representative read quality score, q (right axis). Vertical gray bands demarcate transitions between phases of flight. b Top scoring BLAST results for highest quality “Mars” read, indicated via arrow in a, length 6402. c Start of best match sequence alignment, to J02459.1 Enterobacteria phage lambda, complete genome, length 48502 (range 20562–27113, score 8907 bits(9877), expect 0.0, identities 6108/6651 (92%), gaps 395/6651 (5%), strand Plus/Minus). d Average genomic coverage of lambda for all parabolas based on tombo-aligned bases. Fig. 3 Translocation time is weakly or not affected by vibration. a RMS vibration distributions for ground and flight. b Nanopore translocation time as measured by alignment of ionic current to the genomic reference: distribution for <10 ms. Ground (blue), flight (light brown), both (dark brown). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 24 C.E. Carr et al. smallest mean values of ionic current noise were also observed during hypergravity (0.282; Supplementary Fig. 11). Although statistically larger, the difference between largest mean value for ionic current noise (phase other) was miniscule (0.003). Nanopore sequencing is compatible with many life detection missions from the perspective of mass (~100 g), size, and power (<2 W). Recent work also suggests that MinION electronics and flow cell components would survive radiation doses consistent with life detection missions to Mars, Venus, and Enceladus, although not Europa, without additional shielding . Our work shows that sequencing on all these worlds, including Europa, could be feasible from a g level perspective. In addition, the robustness to vibration suggests that operation concurrent with other mission activities, such as drilling or operation of other instrument payloads, could occur without any substantial negative impacts. In addition, our work highlights the potential for nanopore sequencing on Earth and beyond in mobile and dynamic environments such as on passenger aircraft, drones, wheeled vehicles, ships, buoys, underwater vehicles, or other Fig. 4 RMS vibration and median ionic current noise during flight. platforms. The single 1 s period with median ionic current noise >0.5 has a median absolute deviation (MAD) of >15 and is therefore an outlier (typically defined as MAD >3). METHODS and under altered g levels, including those that would be Acceleration measurement and flight profile segmentation encountered on the surface of Mars, the Moon, and/or Jupiter’s The flight profile was segmented as described in Carr et al. from moon Europa. Due to the limitations of parabolic flight, our zero-g acceleration data collected using a metal-body Slam Stick X™ (Mide Technology Corp.). The accelerometer was mounted next to the MinION to conditions involved mean acceleration around 4× higher (0.041 ± a common baseplate, using double sided sticky tape (3M 950) to provide a 0.005 g) than at the surface of Saturn’s moon Enceladus (0.011 g). near-unity vibration frequency response. Vibrations were measured with Several factors may have influenced the overall quality of our the internal triaxial piezoelectric accelerometer (TE Connectivity Ltd., nanopore sequencing data. The DNA sequencing library used in 832M1) at a frequency of 5 kHz. this experiment was stored for 72 h prior to loading onto the flow cell. As such, the sample was potentially subjected to degradation, Sequencing which could impact read quality, and may have resulted in the loss Sequencing libraries were prepared using DNA derived from Enterobacteria of ligated adaptors. Such conditions would negatively impact the phage lambda (NEB N3011S), fragmented using a g-TUBE™(Covaris® proper loading of DNA into the individual nanopore. In addition, 520079) with the 6 kb protocol. Next, the libraries were prepared using there is an expected degradation of the flow cell over time during the 1D ligation method (SQK-LSK108) using a “one-pot” barcoding sequencing, which could explain some of the time-related trends, protocol and stored at 4 °C for ~72 h prior to the flight. At the time of independent of any effects of vibration or acceleration. Despite storage, the total library DNA was estimated to be 440 ng at 31.4 ng/μlas sequencing for a limited time at any given g level during parabolic assessed by fluorometry (ThermoFisher Qubit® 3.0 Fluorometer with flight, the operation of the MinION for sustained periods on the Qubit™ dsDNA HS Assay Kit, Q32854). ISS gives us confidence that extended periods of reduced g level A flow cell (FLO-MIN106 R9) was loaded on the ground and sequencing does not negatively impact nanopore sequencing. In addition, it performed using an offline version of MinKNOW 1.7.14 in the flight provides confidence in nanopore sequencing as a viable life hardware configuration while the aircraft was on the ground. After 38 min, sequencing was stopped. In flight, sequencing was reinitiated around detection technology in very low but nonzero g environments, 12 min prior to parabolic flight maneuvers, and continued for a total of such as Enceladus. 103 min before termination. After the flight, basecalling was performed Because zero-phase filtering of vibration data effectively with ONT Albacore version 2.3.1 with quality filtering disabled. removed frequencies at or below 10 Hz (Supplementary Figs 5, 6), filtered vibration measurements did not reflect frequencies Sequence data processing where sensor data would be inaccurate due to the non-unity frequency response of the sensor near DC (0 Hz). In addition, this To quantify adaptor sequences, fastq output was trimmed using Porechop (https://github.com/rrwick/Porechop docker container quay.io/biocontainers/ filtering ensured that we could assess the independent effects of porechop:0.2.3_seqan2.1.1-py35_2). Original untrimmed fast5 reads were g level and vibration. aligned to the reference genome (NEB Lambda, equivalent to NCBI The peaks in the vibration spectrum occurs at frequencies NC_001416.1 with mutations 37589 C→T, 45352 G→A, 37742 C→T, and relevant to nanopore sequencing. Despite this, vibration did have 43082 G→A) using tombo (docker container quay.io/biocontainers/ont- any significant impacts on sequence quality nor on ionic current tombo:1.5-py27r351h24bf2e0_0) with the—include-event-stdev option. noise, except during ground-based sequencing, where the explanatory power of vibration was negligible (<1%; Supplemen- Sequencing and acceleration data integration tary Table 7). A custom script was used to parse tombo-processed fast5 files to Random vibration at translocation-relative frequencies could characterize each read and each tombo-aligned genomic base within each exert a minor interfering effect on translocation, although any read (Supplementary Data). A representative read quality score was impact in changes in vibration during flight did not translate into calculated as q ¼10log ðpÞ, where p is the mean of the per-base error p 10 any consistent or large changes in ionic current noise due to the −q/10 5,6 probability p = 10 , where q is the per-base Phred quality score small (0.125 ms) mean difference in translocation times despite estimated via basecalling. Read timings were adjusted by offsets to align nearly a sixfold change in RMS vibration. genomic and accelerometer data (Supplementary Table 2). Each read and Higher g levels tended to be associated with higher read base was assigned one of the following states (parabola, transition, quality, although the effect size is small (Δq = 0.4, hypergravity − hypergravity, other) on the basis of the periods.txt file produced by prior parabola; Supplementary Table 5, an upper bound of ~0.2/g). The analysis and available online at https://osf.io/nk2w4/. npj Microgravity (2020) 24 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA C.E. Carr et al. Vibration data processing Preprint A vibration equivalent to g level (Earth-relative gravity) was computed A previous version of this manuscript was published as a preprint . qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 2 2 as g ¼ g þ g þ g to provide a measure of vibration that is x y z Reporting summary independent of the Slam Stick X™ orientation. The vibration power spectral density (PSD) for g was computed using Further information on research design is available in the Nature Research Welch’s method (MATLAB pwelch() function) with default parameters Reporting Summary linked to this article. (Supplementary Fig. 4). Filtering was then performed for two reasons: (1) to eliminate vibration data where the frequency response of the piezoelectric accelerometer is not unity, and (2) to analyze vibration at frequencies DATA AVAILABILITY related to timescales at which base translocation occurs during nanopore Raw and calibrated data are available via the Open Science Framework at: sequencing, which are overwhelmingly <10 ms (Fig. 3b, Supplementary https://osf.io/n6krq/ and https://osf.io/nk2w4/. Fig. 9). The g level equivalent vibration g was filtered with a high pass infinite impulse response filter (Supplementary Fig. 5) that was generated with MATLAB’s designfilt() function (stopband 5 Hz @ 60 dB attenuation, CODE AVAILABILITY passband 10 Hz with unity ripple, sample rate 5 kHz). Filtering was The MATLAB scripts implementing our analysis are available at: performed using the MATLAB filtfilt() function, which uses forward and https://github.com/CarrCE/zerogseq. reverse filtering to achieve zero-phase delay. The PSD was computed as before for the resulting filtered g level equivalent vibration g (Supple- Received: 10 January 2020; Accepted: 11 August 2020; mentary Fig. 6). RMS vibration was computed in 1 s bins from g using the MATLAB rms() fuction. An overview of vibration is shown in Supplementary Fig. 2 for flight and Supplementary Fig. 3 for ground. Sequencing read quality regression analysis REFERENCES Sequencing read times were adjusted by an offset to place sequencing 1. 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Preprint at https://www.biorxiv.org/content/10.1101/2020.01.09.899716v1. was estimated as the sum of tombo-aligned bases within a given phase of flight divided by the lambda genome size (48,502 bases). Stepwise linear regression, via the MATLAB stepwiselm() function, was used to evaluate ACKNOWLEDGEMENTS the relationship between coverage and parabola period (Supplementary Table 6). We thank the MIT Media Lab Space Exploration Initiative for providing the parabolic flight. This work was supported by NASA award NNX15AF85G. N.C.B. was supported by NASA Postdoctoral Fellowship award 80NSSC17K0688. Base ionic current noise regression analysis A time series of ionic current noise was estimated in 1 s bins by computing the median of ionic current (tombo norm_std output) for all bases within a AUTHOR CONTRIBUTIONS bin. Stepwise linear regression, via the MATLAB stepwiselm() function, was C.E.C. designed the experiment, C.E.C., K.S., S.A.B., and N.C.B. built and tested the used to evaluate the impact of time, RMS vibration, and g level (flight only) hardware, N.C.B. and M.T.Z. collected the data, C.E.C. and N.C.B. processed the data. on median ionic current noise (Supplementary Tables 7, 8). For flight, the G.R. advised on the experiment design. C.E.C. and N.C.B. wrote, and all authors edited regression time was restricted as stated above. and approved, the paper. C.E.C. is the guarantor. Base ionic current noise phase of flight analysis (flight only) CONFLICT OF INTEREST To assess differences in ionic current as a function of phase of flight, we performed a one-way ANOVA via the MATLAB anova1() function on the The authors declare no conflict of interest. non-mux tombo-aligned bases (Supplementary Table 9). To compare group means we then used Tukey’s Honestly Significant Difference test as above. ADDITIONAL INFORMATION Supplementary information is available for this paper at https://doi.org/10.1038/ Does flight vs. ground impact translocation time? s41526-020-00113-9. A Kolmogorov–Smirnov test was performed with the MATLAB kstest2() Correspondence and requests for materials should be addressed to C.E.C. function on the base translocation times for ground vs. flight. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 24 C.E. Carr et al. appropriate credit to the original author(s) and the source, provide a link to the Creative Reprints and permission information is available at http://www.nature.com/ Commons license, and indicate if changes were made. The images or other third party reprints material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims article’s Creative Commons license and your intended use is not permitted by statutory in published maps and institutional affiliations. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give © The Author(s) 2020 npj Microgravity (2020) 24 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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