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ORIGINAL RESEARCH
published: 21 August 2020
doi: 10.3389/fmicb.2020.01909
Validating an Automated Nucleic
Acid Extraction Device for Omics in
Space Using Whole Cell Microbial
Reference Standards
Camilla Urbaniak1†, Season Wong2†, Scott Tighe3†, Arunkumar Arumugam2, Bo Liu2,
Ceth W. Parker1, Jason M. Wood1, Nitin K. Singh1, Dana J. Skorupa4, Brent M. Peyton4,
Ryan Jenson5, Fathi Karouia6, Julie Dragon3 and Kasthuri Venkateswaran1*
1 NASA Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA, United States, 2 AI Biosciences, College
Station, TX, United States, 3 University of Vermont, Burlington, VT, United States, 4 Montana State University, Bozeman, MT,
United States, 5 IRPI LCC, Portland, OR, United States, 6 NASA Ames Research Center, Moffett Field, CA, United States
Edited by:
Rakesh Mogul, NASA has made great strides in the past five years to develop a suite of instruments for
California State Polytechnic University, the International Space Station in order to perform molecular biology in space. However,
Pomona, United States
a key piece of equipment that has been lacking is an instrument that can extract nucleic
Reviewed by:
Nigel Cook, acids from an array of complex human and environmental samples. The Omics in
Jorvik Food & Environmental Virology Space team has developed the µTitan (simulated micro(µ) gravity tested instrument
Ltd., United Kingdom
Tatiana A. Vishnivetskaya, for automated nucleic acid) system capable of automated, streamlined, nucleic acid
The University of Tennessee, extraction that is adapted for use under microgravity. The µTitan system was validated
Knoxville, United States using a whole cell microbial reference (WCMR) standard comprised of a suspension of
*Correspondence: nine bacterial strains, titrated to concentrations that would challenge the performance of
Kasthuri Venkateswaran
kjvenkat@jpl.nasa.gov the instrument, as well as to determine the detection limits for isolating DNA. Quantitative
†These authors have contributed assessment of system performance was measured by comparing instrument input
equally to this work challenge dose vs recovery by Qubit spectrofluorometry, qPCR, Bioanalyzer, and Next
Specialty section: Generation Sequencing. Overall, results indicate that the µTitan system performs equal
This article was submitted to to or greater than a similar commercially available, earth-based, automated nucleic acid
Extreme Microbiology, extraction device. The µTitan system was also tested in Yellowstone National Park (YNP)
a section of the journal
Frontiers in Microbiology with the WCMR, to mimic a remote setting, with limited resources. The performance of
Received: 29 May 2020 the device at YNP was comparable to that in a laboratory setting. Such a portable, field-
Accepted: 21 July 2020 deployable, nucleic extraction system will be valuable for environmental microbiology, as
Published: 21 August 2020
well as in health care diagnostics.
Citation:
Urbaniak C, Wong S, Tighe S, Keywords: International Space Station (ISS), microbial monitoring, automated DNA extraction, microgravity (µg),
Arumugam A, Liu B, Parker CW, extreme environments
Wood JM, Singh NK, Skorupa DJ,
Peyton BM, Jenson R, Karouia F,
Dragon J and Venkateswaran K INTRODUCTION
(2020) Validating an Automated
Nucleic Acid Extraction Device All terrestrial organisms exposed to the environmental conditions of space are subjected to
for Omics in Space Using Whole Cell reduced gravity, high-energy charged particles, high UV levels, low pressure, and large changes
Microbial Reference Standards.
Front. Microbiol. 11:1909. in temperature. When living in such an environment, humans can become immunocompromised
doi: 10.3389/fmicb.2020.01909 (Mehta et al., 2017), microbial behavior can be changed (Kim et al., 2013), and host-microbial
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Urbaniak et al. µTitan for the ISS
interactions can be altered (Foster et al., 2014). To diagnose and to compare this performance against the commercially
patients or to perform microbial monitoring on Earth, samples available, widely used, automated nucleic extraction system,
are immediately sent to a laboratory for testing and results can MaxwellTM. The sensitivity and selectivity of the µTitan system
be obtained in a day or two. However, samples collected from the and how it compared to the MaxwellTM system was assessed
International Space Station (ISS) will take from 2 to 4 months to by Qubit (for DNA yield), qPCR (for 16S rRNA gene copy
return back to Earth before analyses can be performed (Pierson numbers) and the Bioanalyzer (for extracted DNA length). In
et al., 2012), and it would not be practical for future long-term addition, shotgun metagenomics sequencing was performed on
space missions to send samples back to Earth for analysis. To two platforms, Illumina MiSeq and Oxford Nanopore MinION
make exploring and living in space feasible, instruments that in order to assess the ability to produce reliable sequencing data,
automate laboratory analysis will need to be developed. Since in terms of diversity and relative proportions of the bacteria
most molecular diagnostic methods for assessing and monitoring present in the WCMR.
health are based on nucleic acids (Dwivedi et al., 2017), focused
efforts to design portable nucleic acid-based molecular diagnostic
assays (Chan et al., 2016a,c; Priye et al., 2016), compatible with MATERIALS AND METHODS
microgravity, are a high priority.
In 2016, Dr. Kathleen Rubins, a NASA astronaut, became Preparing the Whole Cell Microbial
the first person to sequence DNA on the ISS, using the Oxford Reference Standard
Nanopore MinION sequencer (Castro-Wallace et al., 2017). The whole cell microbial reference (WCMR) standard used to
While this was a great milestone for space molecular biology, validate the µTitan system consists of nine bacteria, five of which
the DNA that was sequenced had already been extracted and are Gram positive and four of which are Gram negative. The
the metagenome libraries prepared on Earth before sending to strains used, their amounts, and other metadata are listed in
space for sequencing (Castro-Wallace et al., 2017). In another Table 1. The WCMR was produced as part of the Association of
flight project, bacterial DNA, which also had been isolated on Biomolecular Resource Facilities (ABRF) metagenomics research
Earth, was sent to the ISS and successfully amplified with the group (Tighe et al., 2017). Briefly, individual bacteria were
miniPCRTM (Boguraev et al., 2017). More recently, methods have cultured on either tryptic soy agar or marine 2216 agar until
been developed to allow for DNA and RNA extraction to occur early log phase growth was achieved, followed by harvesting
on the ISS before in situ downstream analyses. For example, with and resuspension in 1x phosphate buffered saline (PBS) without
the Genes in Space-3 project, cultured bacterial cells that had calcium or magnesium. The cell suspension was washed twice
been isolated from around the ISS were lysed in a thermocycler with 1x PBS followed by vortexing, to ensure that cells were
and the DNA amplified before being sequenced on the MinION in a unicellular suspension and not aggregated (confirmed by
(NASA, 2017). With the WetLab-2 research platform, RNA was microscopy) before the fixation step with ethanol. Fixation was
successfully isolated from Escherichia coli cultures by lysing the performed by adding 100% ethanol in a drop wise fashion while
cells with bead beating and then capturing the released RNA with vortexing until the final concentration was between 90–95%.
the RNAexpressTM column, after which the isolated RNA was Cells were allowed to settle overnight, and the supernatant was
analyzed with qPCR (Parra et al., 2017). The current methods discarded to remove any free-floating DNA. The final sample was
are a tremendous advancement for space genomics because they resuspended in fresh 90% ethanol and stored at 4◦C.
allow for a complete sample-to-analysis process aboard the ISS, Enumeration of each individual ethanol fixed bacterial
but they also have drawbacks. These techniques are manual and suspension was performed by diluting 1 µl of the suspension
take up crew time, are not high throughput, and are better in 48 µl of 1X PBS and 1 µl of 0.1% sodium dodecyl sulfate.
suited for pure cultures rather than complex, mixed microbial Staining was performed by adding 1 µl of Sytox staining reagent,
communities, as they are not efficient for low biomass samples 1 µl of SYBR green (2.5X), 1 µl of enhancer, and 7 µl of buffer
and the nucleic acids are not pure due to the leftover cell (all reagents from Logos BioSystems, Seoul, S. Korea) to this
debris. To address these limitations, the “Omics in Space” team dilution and incubating in the dark at room temperature for
developed µTitan (simulated micro(µ) gravity tested instrument 5 min. Enumeration was performed by adding 4 µl of the stained
for automated nucleic acid extraction), an automated sample cell preparation to a INCYTO counting slide (INCYTO C-Chip
processing system, that is microgravity compatible, and can Hemocytometers, DHC-S025) and counted at 400x magnification
process multiple complex samples simultaneously in an easy to using 485 nm/525 nm epifluorescent microscopy according to
use streamlined manner. Although there are automated sample the manufacturer’s protocol. Combining the individual bacterial
processing devices commercially available for use on the ground preparation into one pool was based on a staggered design. Cell
to process multiple samples simultaneously (e.g., MaxwellTM 16; concentrations ranged from 104 to 106 cells/µl, depending on the
Promega, Madison, MI), these instruments are not microgravity organism (Table 1), resulting in a mixed microbial community
compatible nor are they lightweight and compact. containing 7.3× 106 total cells/µl.
The objectives of this study were two-fold; (i) to develop Before using the WCMR to validate the µTitan system, a
an automated nucleic acid extraction device that would be controlled laboratory extraction of DNA was performed using
microgravity compatible and thus could be used on the ISS, a modified Qiagen procedure by first washing the cells in 1×
and (ii) to validate system performance of this device (i.e., PBS followed by enzymatic digestion of the cell walls with
µTitan) using a whole cell microbial reference (WCMR) standard 20 µl of Metapolyzyme (MPZ) [stock concentration = 10µg/µl]
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TABLE 1 | Metadata of the nine bacteria present in the whole cell microbial reference standard used for validation.
Organism ATCC Gram Genome Reference Volume Cells/µl SD
Number stain size (Mb) Combined
Staphylococcus epidermidis PCI 1200 12228 + 3.5 GCF_000007645.1_ASM764v1 200 µl 1.2 106 1.8 105× ×
Pseudomonas fluorescens F113 13525 − 8.7 GCF_000237065.1_ASM23706v1 150 µl 7.9 105× 1.1 × 105
Escherichia coli K-12 substr. MG1655 700926 − 5.6 GCF_000005845.2_ASM584v2 250 µl 9.9 × 105 1.4 × 105
Chromobacter violaceum NCTC 9757 12472 − 2.7 GCF_000007705.1_ASM770v1 100 µl 2.3 × 106 3.2 × 105
Micrococcus luteus NCTC 2665 4698 + 3.2 GCF_000023205.1_ASM2320v1 50 µl 5.6 5 4× 10 9.5 × 10
Pseudoalteromonas haloplanktis TAC125 35231 − 4.0 GCF_000238355.1_Phal_1.0 300 µl 8.9 104× 5.3 × 103
Bacillus subitilis subsp. subtilis str. 168 23857 + 5.8 GCF_000009045.1_ASM904v1 250 µl 8.3 × 105 1.7 5× 10
Halobacillus halophilus DSM 2266 3567 + 5.6 GCF_000284515.1_ASM28451v1 250 µl 4.0 × 105 5.7 4× 10
Entercoccus faecalis OG1RF 47077 + 3.6 GCF_000007785.1_ASM778v1 200 µl 8.9 × 104 3.8 × 104
(Millipore Sigma, St. Louis, MO, United States) at 35◦C for 4 h, reagent strip is placed inside an enclosure to become a cartridge.
after which DNA extraction was completed using a QIAamp The mechanical movements inside each individual cartridge are
DNA Micro Kit (Qiagen 56304, Germantown, MD) with a enabled by a unique magnetic coupling approach (Figures 2B,C).
pretreatment bead beating step (Matrix A beads MP biomedical These figures show how nucleic acid extraction is performed
116910050CF) to increase lysis efficiency. Measurements from by the stepper motors that are programmed to automatically
a Qubit spectrofluorometer indicated that 3.16 ng of DNA was control the movement of the external magnets, which in turn
obtained per 106 cells. drive the movement of the components inside the cartridge. The
“driver magnets” outside of the cartridge precisely control the
Overview of µTitan movement of “follower magnets” within the cartridge to process
The µTitan system is based on a fused deposition modeling 3D nucleic extraction and elution. The two sets of magnetic particles
printer in which the motion and temperature controls have been (MPs) are held together by the magnetic field that penetrates
repurposed for automated NA extraction, with the capability through the cartridge’s plastic wall. Synchronized motions of
to perform both heated incubation and NA amplification the magnetic tip-combs enable reproducible and controllable
(Figures 1A,B). The system is portable and robust and was able action inside each cartridge, including MP mixing. Figure 2D
to perform extractions while being transported in the back of shows the process of effective mixing and washing of MPs. These
a sports utility vehicle (SUV) at highway speed (Figure 1C). external “driver magnets” allow for precise positioning of the
The extraction efficiency of Chlamydia trachomatis DNA from “MP manipulating tip” located inside the cartridge. The bottom
urine samples using an early prototype was demonstrated to be pair of magnets below the wells leads to release and capture of
comparable to the spin column method from Qiagen (Chan et al., the MPs by this tip, allowing the MPs to move throughout the
2016a). Extractions of RNA from the Zika virus in urine and wash buffer, allowing for effective mixing and washing of these
saliva samples also proved to be successful (Chan et al., 2016b). MPs. Under normal gravity, the magnet inside the tip falls back
down when the bottom repelling magnet is removed (Figure 3A),
Mechanics of µTitan Designed for Use but this is not the case in reduced gravity as the magnet inside
on the ISS the tip will just remain “floating” inside (Figure 3B). For the
The current version of µTitan is 34 34 36 cm when encased microgravity adapted machine, a set of opposing field magnets× ×
with a cover. It weighs about 8 kg including the power adaptor. was installed to pull the extraction tip magnet down (Figure 2D).
A laptop is currently used to operate the device, but the These innovative steps allow the elimination of pipetting in
protocol program can be saved on a secure digital (SD) card if space under microgravity, which is difficult, laborious, and often
needed. A comparison between the MaxwellTM and the Titan results in mishandling.µ
specifications are presented in Table 2.
Individually enclosed environments for sample extraction are DNA Extraction Using µTitan and
typically used to prevent cross-contamination. This is essential MaxwellTM
in the ISS environment to avoid the unintentional release of Pre-processing
extraction reagents and potentially harmful microbes. Currently, One milliliter of the WCMR was pre-processed by incubating
µTitan uses aluminum-foil-sealed, pre-filled reagent strips placed with 48 µl of 10 µg/µl MPZ for 15 min at 35◦C, followed by
inside a strip holder that can be closed after sample input 15 s of bead beating with Matrix Lysing E bead beating tubes
(Figure 1D). Using this approach, magnetic coupling can be (Mp Biomedicals) using a battery-powered oscillating power tool
used to perform extraction with multiple mounted cartridges. (Ryobi JobPLUS ONE 18V multi tool with P246 console & P570
The operation of the coupling mechanism is similar to that of a Head attachment). The sample tube was placed and fixed tightly
magnetic fish tank cleaner, where the motion of NA extraction on the flat end of the blade using blue painters’ tape. In YNP,
can be controlled inside the cartridge without direct contact the WCMR was first subjected to bead beating (as above), then
from outside. Figure 2A shows the basic set-up where an 8-well incubated with 25 µl MPZ for 1 h at 37◦C, followed by another
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FIGURE 1 | Concept of converting a low-cost 3D printer to perform rapid and automated NA isolation. (A) A typical FMD type 3D printer. (B) A 3D printer turned
extraction device. The extruder of a 3D printer was removed to allow the adaptors to be mounted. The magnetic particle processing tip-comb (tip-comb) was
attached on the mount. Its vertical and lateral movements are controlled by two Z-motors and one upper X-motor controls. Another X-motor control below the
extraction cartridges enable resuspension of magnetic particles for NA binding and washing (see description later). The tip-comb has 6 fingers with each finger has
magnets to use magnetic coupling to externally control the extraction tips inside an extraction cartridge to perform extraction. There is no direct contact between the
magnets, the samples, and MPs. Six samples can be processed simultaneously under the same protocol program. The heated strip under the elution wells uses the
3D printer’s extruder heater and thermistor to provide precise temperature control for heated NA elution. (C) µTitan placed inside the cargo area of an SUV. (D) A
reagent strip that has been pre-filled with the appropriate extraction reagents.
round of bead beating. Next, 100 µl of the pre-processed sample correspond to an approximate input of 107 cells, while the
was added to either the MaxwellTM or µTitan system. The high low biomass samples (Tx_Max_Low, Tx_µT_Low) correspond
biomass samples (Tx_Max_High, Tx_µT_High, YNP_µT_High) to an approximate input of 104 cells. The “medium” biomass
samples (YNP_µT_Med) corresponds to an approximate input
of 106 cells.
TABLE 2 | Specifications of the µTitan and MaxwellTM systems evaluated in this
study.
µTitan Extraction
MaxwellTM µTitan A commercially available magnetic particle–based NA isolation
# samples run simultaneously 16 6 kit (NucliSENS Magnetic Particle Extraction Kit, bioMerieux,
Dimensions (W × D × H) 33 × 44 × 33 cm 34 × 34 × 36 cm Durham, NC) was purchased, and the reagents from this kit
Weight 18.9 kg 8.0 kg were used to pre-fill the µTitan cartridges. These reagents
Typical protocol duration 37–40 min 10–20 min included lysis buffer, magnetic particle solution, wash buffer #1,
Enclosed cartridge No Yes wash buffer #2, wash buffer #3, and elution buffer. The µTitan
Heated elution Yes Yes extraction protocol (e.g., volume, time of incubation, and number
Programmable protocols Yes Yes of repeated washing steps) is as follows: 100 µL of the pre-
Power requirements 100–240VAC, Power adaptor: processed sample was added to the second well of a µTitan
50–60Hz, 2.1A 100–240VAC,50–60Hz,
1.8A Output to 12V cartridge containing 400 µL of lysis buffer and incubated for
and 10A 10 min. Eight microliters of NucliSENS magnetic particles, for
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FIGURE 2 | Extraction cartridge, magnetic coupling, and schematic of sample extraction. (A–C) External magnetic control and movement of sample by magnetic
particles outside of extraction cartridge. (D) Detailed overview of magnetic handling, washing, and elution of NAs within the µTitan cartridge. The magnetic tip can
move freely on the wall of the cartridge as actuated by the driver and follower magnets. The placement and the polarity of the magnets inside the extraction tip and
those underneath the wells enable mixing and washing.
capturing NAs, were then added to the lysed sample solution in temperature, and amount of material to be deposited. Since this is
the second well. The magnetic particles were intermittently mixed pre-programmed, a user just needs to click on the “print” button
for 10 min, allowing for NA to bind to the MPs. The cartridge was icon to start the automated extraction process.
then secured on the µTitan system. An extraction tip mounted Once the program started, the extraction tip comb (driver)
on the follower magnet set was placed on the first well of the magnet picked up the extraction tip magnet inside the cartridge
cartridge. Wells # 3–7 contained wash buffer # 1 (600 µL and box using a magnetic coupling mechanism and moved it to
250 µL), wash buffer # 2 (600 µL and 250 µL), and wash buffer # 3 the subsequent wells of the cartridge. The NA bound magnetic
(250 µL). Well # 8 contained an elution buffer (60 µL). Each pre- particles attached to the base of the extraction tip were then
processed sample was run in triplicate. To ensure the quality of washed sequentially in wash buffer # 1 and # 2, twice for 2 min
the work and to check for contamination, molecular-grade water each, and wash buffer # 3 for 30 s. Next, the magnetic particles
was used for extraction during each run of the machine, instead were air-dried for 4 min. When the air-drying process began, the
of sample, and is considered the machine control (“Tx_µT_CTL” heater strip at the bottom of the elution well of the cartridge
or “YNP_µT_CTL”). was heated to bring the elution buffer temperature to ∼70◦C
The µTitan system was operated using a laptop computer by a cartridge heater commonly used as the extruder of a 3D
(connected with a USB cable), loaded with an open-source printer. This extruder heater’s function was also controlled by
software called Repetier-Host (Hot-World GmbH & Co., Willich, the program, and the temperature was precisely monitored with
Germany) that uses G-code based programming commonly used a thermistor. The NAs captured on the magnetic particles were
for 3D printing and CNC milling. The G-code written by AI eluted for 5 min in 60 µL of elution buffer. After elution, the
Biosciences for µTitan, was based on that written for a standard magnetic particles were captured again by the extraction tip and
3D printer, which provides instructions on the movement, moved back to well # 1, leaving only the eluate in the elution
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FIGURE 3 | The µTitan cartridge at 1g vs reduced gravity. (A) Magnetic particle extraction in 1-g condition. Magnet inside of the extraction tip will fall back down to
the bottom of the tip after the magnet with opposite polarity is removed. (B) In reduced gravity, the repelled extraction tip will remain afloat even after the bottom
magnet is removed. Therefore, the paired-magnets mentioned in Figure SW3D were used.
well (well # 8). The eluate was collected and stored in a 500 µL purified DNA from both machines was stored at ◦−20 C until
tube for future analysis. The µTitan system has the capability of further analysis.
processing 6 samples in parallel.
Qubit Quantification and Quantitative
PCR (qPCR)
MaxwellTM Processing Samples were quantified using two methods: the Q32854
Parallel extractions of the pre-processed samples were carried QubitTM dsDNA HS Assay (ThermoFisher, Waltham, MA,
out on the MaxwellTM automated extraction system (Promega United States) and qPCR using a standard curve SYBR green
Corporation, Madison, WI, United States) using a DNA kit method using two control DNA standards. Qubit assay was
(AS1520) and the “MaxwellTM RSC blood DNA” run program. performed using a 2 µl aliquot of DNA mixed with 198 µl of the
Briefly, 100 µL of the preprocessed samples were added to the dsDNA HS reagent and read on a Qubit 4 spectrofluorometer.
cartridge and placed on the cartridge holder tray, and the DNA qPCR was performed on replicate 1 µl samples of 1:10
was eluted in 60 µL of elution buffer. Each pre-processed sample dilution of test DNA using the PerfeCTa SYBR Green SuperMix
was run in triplicate. To ensure the quality of the work and to (95054 Quanta Bio, Beverly, MA, United States) using primers
check for contamination, molecular-grade water was used for recommended by the Earth Microbiome Project -515 Forward
extraction during each run of the machine, instead of sample, GTGYCAGCMGCCGCGGTAA (Parada et al., 2016) and 806
and is considered the machine control (“Tx_Max_CTL”). The Reverse GGACTACNVGGGTWTCTAAT (Walters et al., 2016).
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FIGURE 4 | DNA quantification of the extracted WCMR. DNA extraction of the WCMR was performed by both MaxwellTM and µTitan systems in the lab of AI
Biosciences (Texas; “Tx”) or in the field by µTitan (Yellowstone National Park; “YNP”). The qPCR Ct values are shown by the blue bars. Known amounts of WCMR
DNA, extracted by Qiagen, was used to generate a standard curve during qPCR, by which DNA yield in ng could be calculated (purple bars). NB: The lower the Ct
value, the higher the amount of DNA in that sample. The DNA yield was also measured by Qubit (gray bars). Different amounts of biomass; high, medium, or low
were extracted with both MaxwellTM and µTitan systems. High biomass samples represent inputs of approximately 107 cells, low approximately 104, and medium
approximately 106. As can be observed, the µTitan system generated higher DNA yield than the MaxwellTM system for both high and low biomass samples. Also,
the µTitan system in the field produced comparable results to that obtained in the lab (YNP_µT_High vs Tx_µT_High).
Standard curves were generated from two separate microbial (95◦C-15s| 60◦C-15s| 95◦C-15s). Primers were included at a final
reference control DNA, including (1) purified genomic DNA concentration of 0.1 µM and low ROX as an internal reference.
(gDNA) from the WCMR that had been extracted with Qiagen
and (2) The ABRF/ATCC©R MSA-3001 gDNA (ATCC, Manassas,
VA). Both standard curves were generated from 2, 0.2, 0.02, 0.002, DNA Molecular Weight Analysis
and 0.0002 ng/µl of DNA. qPCR thermocycling was performed in (Fragment Length) Using Bioanalyzer
25 µl reactions on an ABI 7900HT (Applied Biosystems, Foster DNA fragment size analysis (molecular weight) of the extracted
City, CA, United States) using the following three-step program: DNA was evaluated using the Agilent Bioanalyzer 2100 with
Denatured at 95◦C -3 min, 40 cycles of PCR (95◦C -15 s| 54◦C- the High Sensitivity DNA chip (5067-4627) and the Advanced
60 s| 72◦C -60 s), followed by a standard dissociation curve Analytical Technologies Fragment Analyzer 5200 with the HS
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FIGURE 5 | Relative abundances of the nine bacteria present in the WCMR. The WCMR DNA that was extracted by the MaxwellTM and µTitan systems was
sequenced on the Illumina HiSeq platform, and the relative abundances of the nine bacteria present in the WCMR shown in this bar graph. Each bar represents a
sample, and each colored box represents the proportion of a particular organism in that sample. For comparison, the WCMR was also manually extracted with
Qiagen, sequenced, and plotted. The high and medium biomass samples were able to detect all nine organisms, in the same relative abundances, which was also
comparable to that observed with Qiagen extraction. The low biomass samples did not detect all bacteria in the WCMR standard, but the µTitan system detected
more than the MaxwellTM. A complete list of the bacteria detected and their counts are summarized in Table 3.
Large Fragment 50kb Kit (DNF-493-0500) according to the Metagenome Sequence Data Processing
manufacturer’s instructions. Adapter sequences and low-quality reads (Phred score < 20
across the entire length of the read) were removed with
Illumina Sequencing Trimmomatic and reads that were shorter than 80 bp after quality
Library Preparation control trimming were discarded. Post-processing resulted in
Whole genome shotgun sequencing libraries were synthesized 53,549,976 high-quality reads. DIAMOND (Buchfink et al.,
using Nextera XT reagents (Illumina Corp, San Diego, 2015) and the weighted lowest common ancestor (LCA)
CA, United States) from either 3 ng of total DNA for algorithm of MEGAN6 (Huson et al., 2007) (Settings of
samples > 0.5 ng/µl or 6 µl input for samples below 0.4 ng/µl. minScore = 50, maxExpected = 0.01, topPercent = 10, and
Final libraries were assessed for quality using the QubitTM minSupportPercent = 0.01) was used to cluster high-quality
dsDNA HS Assay and Agilent Bioanalyzer 2100 assessment using filtered reads to taxonomic and functional levels. BLAST hits
the DNA high sensitivity chip (5067-4626 Agilent Corp., Santa of ≥ 20 amino acids and ≥ 90% similarity were collected and
Clara, CA, United States). All samples, including positive and used for taxonomic and functional assignment using the NCBI
negative controls, were pooled by combining the high input taxonomy database which contains over 6.6 105× reference
(>0.5 ng/µl), using 2.5 ng of library, with the low input, using sequences (Sayers et al., 2009), and NCBI-NR protein sequence
0.25 µl, to prevent over representation of low input samples. database which consists of entries from GenPept, SwissProt,
Pooled hybridization cocktails were clustered and sequenced PIR, PDB, and RefSeq, were used with MEGAN6 (Huson et al.,
using a rapid run SR flow cell (GD-402-4002) for 150 bases on 2007) to perform the taxonomic and functional binning of the
the Illumina HiSeq 1500. metagenomic reads.
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Oxford Nanopore Sequencing
Oxford Nanopore libraries were synthesized for all samples from
input ranges of BDL (below detection limit of the Qubit) up to
5 ng for higher input samples using the Rapid PCR barcoding
kit (RPB004 Oxford Nanopore Technologies, Oxford, United
Kingdom) with the following modifications: high input samples
were amplified using the standard method of 14 PCR cycles,
while samples that were below the detection limit of Qubit
were amplified using 22 PCR cycles. Barcoded libraries were
pooled equally at 1 fmol each for the high samples, while the
BDL samples were pooled at the maximum volume allowable
for library input. Sequencing was performed using the MK1B
MinIon with 9.4 flow cell yielding > 1 million reads of 2 Gbases
with average read lengths of 2–8 kb. All quality control statistics
were within specification as outlined by Oxford Nanopore.
Microbial reference controls were also sequenced on the
Oxford Nanopore sequencer, including the purified gDNA from
the Qiagen Benchmark sample. Rapid PCR barcoded libraries
were synthesized from input concentration identical to the
qPCR standard curve at 6, 0.6, 0.06, 0.006 ng. This titration
was performed to determine the detection limits, linearity, and
efficiency for ultra-low input samples.
Oxford Nanopore data was analyzed using the WIMP (What’s
In My Pot) module of the EPI2ME software (Oxford Nanopore
Technologies, Oxford, United Kingdom), and raw read count
data was exported as CSV data for manual parsing and
enumeration using Microsoft Excel.
Nanopore detection and sequencing of the low biomass
samples revealed negligible concentrations above background.
This is not surprising since the input concentrations were below
the recommended input for the Rapid PCR barcoding kit.
For these reasons, Nanopore data generated from low biomass
samples were not included.
Data Analysis
The Illumina sequencing data was analyzed in R version 3.3.1
using the Hmisc, ggplot, compositions and cluster packages. For
the PCA plot, the data was first center log ratio (clr) transformed
and the Euclidian distances plotted. K-means clustering was used
to determine the number of distinct groups and the samples that
belonged to these groups.
DNA yield and qPCR graphs were generated in Prism version
7.0. Statistical analyses were executed using one-way analysis of
variance (ANOVA) and then using the Benjamini- Hochberg
false discovery rate (FDR) multiple test correction. Statistical
significance was set at P < 0.05.
RESULTS
Testing and Validation of the µTitan
System
A whole cell microbial reference standard, that contained intact
microbial cells (Table 1) was extracted using both µTitan and
MaxwellTM instruments in a laboratory setting. High biomass
(107 cells) and low biomass (104) cellular concentrations were
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TABLE 3 | Illumina sequencing read counts of the bacterial species present in the whole cell microbial reference standard detected from the µTitan and MaxwellTM systems in both a laboratory and field setting.
Microbial taxa # sequences retrieved with high biomass # sequences retrieved with low biomass # sequences retrieved with high and medium
samples in a lab (Texas) samples in a lab (Texas) biomass samples processed with µTitan in the
field (YNP)
MaxwellTM (Tx_Max_High) µTitan (Tx_µT_High) MaxwellTM (Tx_Max_Low) µTitan (Tx_µT_low) YNP_µT_High YNP_µT_Medium
rep 1 rep2 rep3 rep1 rep2 rep3 rep1 rep2 rep3 rep1 rep2 rep3 rep1 rep2 rep3 rep1 rep2 rep3
Escherichia coli 2,88,592 3,17,753 3,26,481 2,81,112 2,79,525 2,73,380 1,027 421 561 1,955 1,310 2,171 3,18,603 3,34,962 3,61,581 2,42,269 2,50,667 2,70,935
Chromobacterium 2,22,242 1,82,297 1,67,347 1,26,575 1,27,509 1,19,165 465 243 350 1,255 852 1,162 1,49,285 1,64,426 1,73,854 1,23,261 1,60,574 1,13,173
violaceum
Enterococcus 27,762 34,981 48,527 79,526 74,161 75,180 641 274 391 2,458 1,278 3,034 99,057 1,07,369 1,22,557 61,701 53,609 1,27,570
faecalis
Bacillus subtilis 26,060 31,835 38,835 44,318 43,126 43,181 – – – 426 301 496 59,178 58,916 66,009 44,056 36,867 81,776
Pseudomonas 38,503 37,911 35,184 27,282 26,703 26,217 – – – 120 – – 32,041 33,889 35,996 23,954 27,818 23,405
fluorescens
Micrococcus luteus 56,593 38,511 34,463 26,957 26,382 24,319 – – – 218 161 210 19,898 21,191 21,476 14,784 21,192 10,843
Staphylococcus 17,675 23,449 24,162 25,813 22,314 24,302 – – – – – 178 22,921 20,992 21,141 10,689 6,422 20,576
epidermidis
Pseudoalteromonas – 2,793 2,873 2,474 2,399 2,500 – – – – – – 3,227 3,213 3,503 2,234 – 3,492
sp.
Halobacillus sp. 2706 3298 3182 2775 2621 2618 – – – – – – 3590 3754 4177 2341 1966 3132
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FIGURE 6 | Principal coordinate analysis of shotgun metagenomic sequencing data generated from the Illumina platform. Results for each sample were plotted on a
3-D, 3-axis plane, representing 73% of the variation observed amongst all samples. Each point represents a sample, and the closer a sample is to another, the more
similar their microbiome composition (diversity and abundance). There were 5 distinct groups in this dataset, represented by the colored ellipses, which were
determined by unsupervised k-means clustering.
used in the validation tests. WCMR standard was extracted using system had increased performance over the MaxwellTM and
both the µTitan and MaxwellTM instruments in a laboratory from the DNA extracted, six out of nine bacteria were identified
setting. The composition of the WCMR (Table 1) contains (“Tx_µT_Low”), while from the MaxwellTM extracted DNA, only
fully intact microbial cells of Gram +/−, high and low GC, three out of nine bacteria were identified (“Tx_Max_Low”). The
and a range of morphologies. Two cellular concentrations proportions of identified bacteria in the high biomass samples
were used in the extraction tests of the two instruments were similar between the two instruments as well as for the
representing high biomass (107 cells) and low biomass (104 Qiagen Benchmarking Control (Figure 5).
cells) inputs and subject to pretreatment with a cell wall The µTitan system was further evaluated in a Phase II study
digesting enzyme mix (Metapolyenzyme) and mechanical bead at Yellowstone National Park (YNP) to test its performance in
beating. After pretreatment, replicate 100 µl aliquots were a remote setting with limited resources. The MaxwellTM system
used as inputs to both instruments. Following extraction, was not included in this field study due to its size, weight,
the yield of recovered DNA was determined using a Qubit power, and calibration requirements. In this phase of testing, two
spectrofluorometer and qPCR (Figure 4). These results indicate concentrations of the WCMR were used; a high biomass sample
that the µTitan system produced higher DNA yields, with both (“YNP_µT_High”) similar to that used in the laboratory test
the high biomass (“Tx_x_High”) and low biomass (“Tx_x_Low”) (“Tx_µT_High”) and a medium biomass sample composed of a
samples; however, only the high cellular concentrations were 10-fold dilution of the high biomass sample (“YNP_µT_Med”,
statistically different. 106 cells).
Shotgun metagenomic sequencing was performed on all The resulting DNA yield for the high biomass sample, as
samples using the Illumina HiSeq 2500 platform to determine measured by qPCR was comparable to the yield obtained in the
whether the isolated DNA could be used to prepare successful laboratory tests (“Tx”), indicating the µTitan system is able to
sequencing libraries and obtain high-quality sequencing results. produce consistent results even when used in a remote setting
Table 3 shows the WCMR members detected in each sample (Figure 4). Results of the Illumina-based shotgun metagenomic
from both extraction instruments and their respective counts. sequencing were also consistent with the laboratory tests in that
The bar graph in Figure 5 shows the proportion of these nine all nine bacteria in the WCMR were detected in both the high
bacteria in each sample and also includes the proportion of and medium biomass samples (Table 3), in the same proportions
bacteria detected when a manual Qiagen QIAquick extraction as that observed in the “Tx_µT_High” and Qiagen benchmark
(Qiagen Benchmarking Control) was performed in the lab with sample (Figure 5).
high efficiency pre-processing steps added. Both Table 3 and As is expected from any shotgun metagenomics sequencing
Figure 5 show that DNA from all nine bacteria could be run, there will be some measure of contaminant reads that come
isolated from the high biomass samples extracted with either from extraction and sequencing processes (the “kitome”), and
the µTitan (“Tx_µT_High”) or the MaxwellTM instrument this dataset was no exception. The heatmap in Supplementary
(“Tx_Max_High”). For the low biomass samples, the µTitan Figure S1 shows the bacterial contaminants (i.e., any species that
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TABLE 4 | Enumeration and detection of genera belonging to the whole cell microbial reference control using Oxford Nanopore Sequencing.
Microbial taxa Number of reads for high biomass Number of reads for samples Benchmark control: DNA
samples (Texas lab trials) extracted using the µTitan in the extracted with Qiagen
remote field location (YNP)
MaxwellTM µTitan YNP_µ YNP_µ Benchmark DNA Control
(Tx_Max_High) (Tx_µ T_High) T_High T_Medium DNA (ng Input)
rep 1 rep2 rep3 rep1 rep2 rep3 rep1 rep2 rep3 rep1 rep2 rep3 6.0 0.6 0.06 0.006
Escherichia 17454 16576 17,445 12,727 13,265 13,598 15442 14494 15710 18035 10155 18087 17420 10799 658 20
Chromobacterium 1998 1280 1,651 941 1510 1051 1085 1090 1447 1223 2501 1216 1618 1810 121 0
Enterococcus 918 1015 1,002 2766 3024 3063 2726 3339 2762 1370 2808 1378 2132 2991 142 11
Bacillus 4659 4883 5,148 5715 6989 5757 8440 8465 8138 8828 8942 8201 8823 8841 434 21
Pseudomonas 7428 4730 6,051 3068 4336 3470 3728 3545 4818 6861 6829 7543 7259 6770 585 35
Micrococcus 84 73 96 28 62 28 17 18 29 18 123 26 46 50 1 0
Staphylococcus 6555 10459 8,060 9891 9280 13032 5114 6098 5050 3289 5344 3727 2553 2843 173 11
Pseudoalteromonas 5850 6471 6,006 6401 6918 6694 9501 9123 8029 5880 7770 5304 6479 7745 350 19
Halobacillus 105 116 113 125 104 111 125 101 89 99 68 109 78 119 2 0
Data normalized to 50,000 reads for sample.
FIGURE 7 | Comparison of Illumina Nextera XT data to Oxford Nanopore Rapid PCR barcoding data. Percent abundance of the WCMR bacteria detected by either
Illumina short read versus Oxford Nanopore long reads, from DNA isolated from either the MaxwellTM or µTitan system were compared with boxplots. The height of
the box represents the average of triplicate DNA extractions from just the high biomass samples. The error bars represent the standard deviation. Generally, for high
biomass samples, the µTitan system performs as well as the MaxwellTM, and in this case, Illumina short reads worked better for certain genera (Chromobacterium,
Enterococcus, and Micrococcus), while the Nanopore long reads worked better for others (Pseudomonas, Pseudoaltomonas, Bacillus, and Staphylococcus).
did not belong to one of the nine bacteria in the WCMR along system (green points) or with the MaxwellTM instrument (blue
with their counts) in all samples. points), and were distinct from the kitomes and machine
A PCA plot (Figure 6) comparing the diversity and relative controls. With the low biomass samples, the µTitan samples
abundances of the species present in the samples and controls (Tx_µT_Low, pink points) were distinct from their respective
(both WCMR bacteria and contaminants) showed that the controls, but this was not the case with the MaxwellTM samples
high and medium biomass samples had similar microbiomes, (Tx_Max_Low, purple points), which were indistinguishable
regardless of whether they were extracted with the µTitan from their respective controls. Additionally, the PCA plot
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TABLE 5 | Fragment length and molecular weight results for instrument extracted samples and reference DNAs using the Agilent Bioanalyzer 2100.
DNA Orgin Replicate Range (MW) Mean Average
Low High Low High Mean
Tx_Max_H 1 527 19642 5484
2 580 15785 5817 581 17324 5604
3 636 16544 5511
Tx_µT_H 1 510 20213 6337
2 543 27570 6463 534 24126 6405
3 550 24595 6415
YNP_µT_H 1 200 23683 6683
2 527 29271 6977 498 25187 6845
3 766 22608 6876
YNP_µT_Medium 1 1035 22040 9412
2 1142 19653 9058 1060 23274 9341
3 1003 28131 9554
Qiagen extracted WCMR 1 191 41703 8025
2 312 34104 7924 234 34924 7912
3 200 28965 7788
indicates that replicate extractions with the µTitan system were The proportions of the WCMR were compared between
consistent and produced comparable results (replicates group Illumina and Nanopore data for the purpose of detecting any
together on the plot). More importantly, the plot shows that the bias inherent to the sequencing method (Figure 7). These data
field-operated µTitan system (YNP_µT_High) and lab-operated indicates that Illumina short-read sequencing using Nextera XT
system (Tx_µT_High) produced similar results. libraries has increased ratios of detection for Chromobacterium,
In addition to the Illumina HiSeq data, shotgun sequencing Enterococcus, and Micrococcus, while the Oxford Nanopore rapid
was also performed on the high and medium biomass samples PCR data has an increase detection ratio for Pseudomonas,
using Oxford Nanopore Technologies’ MinIon MK1B. The Pseudoalteromonas, Bacillus, and Staphylococcus.
importance of including Nanopore data in this study was The extracted DNA from the high and medium biomass
to provide an alternative sequencing technique that would samples were also analyzed using an Agilent Bioanalyzer 2100 to
avoid any Illumina Nextera short-read sequencing biases due determine the DNA fragment length. For both the MaxwellTM
to GC content, to enable detection using long reads with and µTitan instruments, the fragment length amongst the
an alternative library synthesis that may influence taxonomic replicates were very consistent, with the µTitan system producing
classification due to genome complexity and long genomic slightly larger DNA fragments (average length: 6,625 bp)
repeats (e.g., Pseudoaltermonas), and to determine how DNA compared to the MaxwellTM system (average length: 5,604 bp).
extracted from µTitan would sequence on the MinION MK1B These results were confirmed with a subsequent analysis
as this is the sequencing platform that is currently being used using a high-sensitivity assay with the Advanced Analytical
on the ISS and in the field. Although read depth, read length, Fragment Analyzer 5200 (data not shown), which was closer
and Q-Score are different between Nanopore and Illumina to the fragment length obtained by using a Qiagen Benchmark
sequencing technologies, the organisms belonging to the WCMR control manual extraction (average length: 7,912 bp) (Table 5;
were just as easily detected and classified with the MinION Supplementary Figure S2).
using DNA isolated from both the MaxwellTM and µTitan
systems, in both the laboratory and field settings. Tabulation
of organisms in each sample belonging to the WCMR was Microgravity Compatibility Testing of the
represented at the Genus level on raw data normalized to 50,000 µTitan System
reads to allow for direct comparison between samples (Table 4). While the µTitan cartridges used in these experiments were
These results indicate that, when compared to the MaxwellTM not designed for microgravity compatibility, the newest set
instrument, the µTitan system generated a marginally greater of cartridges have been designed to exploit surface tension
number of reads of Staphylococcus and Bacillus, and an even properties to prevent the unwanted release of fluids (Weislogel
greater number of Enterococcus, all of which are Gram-positive et al., 2009, 2011). These cartridges accommodate fluids and
bacteria. On the other hand, more reads were observed by a magnetic probe and allow the fluids to preferentially remain
the MaxwellTM instrument for the Gram-negative bacteria, at the base of the cartridge well, discouraging wetting of the
Escherichia and Pseudomonas. These trends observed with upper portion of the well, thus preventing liquids from floating
the Nanopore data are consistent with that observed with free when used in microgravity (Weislogel et al., 2009, 2011).
the Illumina data. The microgravity fluid physics behind this design comes from
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Urbaniak et al. µTitan for the ISS
the same team who engineered the “space cup” in 2015, which types (i.e., environmental or human) of complex microbial
allowed astronauts on the ISS to drink hot espresso from an open communities, for both high biomass and low biomass input,
coffee cup for the first time (NASA, 2015). Several 2.1s drop and not only for high concentration of pure bacterial cultures,
tower tests have been performed on the µTitan system (used which the previous methods were tested on. The µTitan system
in the current study) and the newly designed cartridges, and is also automated and requires less crew time than previous
during these low-gravity tests, the fluids remained at the bottom protocols. The dedicated clean-up and washing steps allow for
of the cartridge wells, as designed (Supplementary Figure S3), cell debris to be removed, leading to higher purity nucleic acids,
indicating its compatibility with use on the ISS. allowing for better sensitivity for low biomass samples and overall
better detection of the diversity within a sample. Most current
automated systems on the market, as well a manual extraction
DISCUSSION kits suggest the inclusion of an optional bead beating step if
the user so desires. In addition to bead beating our team also
This manuscript describes the development and validation included the use of metapolyenzyme (MPZ), as it has been
of the µTitan system, a high-performance, automated, and shown for even greater lysis efficiency than just bead beating
programmable nucleic acid extraction platform designed for use alone. The reagents within the µTitan system would be able
in both microgravity and Earth-based assays. The system employs to extract DNA without bead beating and MPZ treatment but
the use of magnetic beads, a probe, and specialized chemistry for low biomass samples, when one needs to extract as much
for the isolation of high-quality nucleic acids for downstream DNA as possible, these additional lysis steps are important.
analyses such as qPCR and NGS. This has all been made possible An additional pre-processing module is being developed where
by the low cost and high precision of consumer-level 3D printers enzymatic digestion and bead-beating will be performed which
(Coakley and Hurt, 2016). will be compatible for the µTitan system and will be in an
The results of the present study have shown that the µTitan enclosed system to prevent aerosols on the ISS. The µTitan
system can isolate high molecular weight DNA that can be system is useful for most the microorganisms but for hardy
used for qPCR and high-quality NGS libraries for both Illumina microorganisms such as spore-formers and actinobacteria, a pre-
and Oxford Nanopore platforms regardless of the assay. The processing step is needed and thus has been included in the
instrument run time could extract high-quality DNA from the design, if needed.
WCMR in 20 min (µTitan) compared to 40 min (MaxwellTM) NASA performs routine microbial monitoring on the ISS, but
with the comparative method used in this study. Additionally, the culture-based methods that are currently used do not provide
the system produced higher DNA yields (Qubit and qPCR) a comprehensive assessment of what microbes are actually
and slightly higher molecule weight DNA fragments (Agilent present. For this reason, NASA is actively working toward
Bioanalyzer) than the MaxwellTM system. Both the Illumina increasing their monitoring capabilities by testing different
and Nanopore sequencing platforms showed that the µTitan in situ protocols for DNA extraction that can be used for
system is able to isolate DNA from Gram-positive organisms downstream qPCR on the ISS (Cherie et al., 2013). The
better than the MaxwellTM system. The µTitan system also advantage of the µTitan system over the other methods currently
detected more WCMR bacterial species in the low biomass being tested is that, unlike a minimum of 105 cells per µL
input and was also less influenced by processing and sequencing that is needed to positively detect microorganisms, a µTitan
contaminants than the MaxwellTM system. The potential for cross output concentration of 200 cells/µl is able to be successfully
contamination when multiple samples are run simultaneously analyzed by NGS and qPCR because of the chemical processes
was also examined. Using both high biomass and low biomass involved in the extraction workflow that increase DNA yield and
samples as input and running them alongside negative control purity. Furthermore, high-quality DNA from six samples can be
samples (i.e., water as the input), no evidence of carryover from obtained in 45 min, with only 15 min of crew time, allowing for
sample to negative control was observed when tested with Qubit high-throughput sample processing aboard the ISS as opposed to
and qPCR (data not shown). manual, laborious, and time-consuming pre-existing methods.
While great strides have been made by NASA in the past five The ability to perform high-throughput sample processing
years to allow for molecular biology to be performed on the onboard the ISS followed by NGS makes it possible for
ISS, with WetLab-2 (Parra et al., 2017), miniPCRTM (Boguraev astronauts to know what microbes and their properties are in
et al., 2017), Razor-EX (Cherie et al., 2013), and MinION (Castro- the environment, at any given time. This is significant when
Wallace et al., 2017), there exists a need for an automated, you consider that omics analyses on ISS samples (performed on
multiple sample processing system for the ISS for downstream Earth) have detected novel organisms and/or those with unique
omics analyses. The development of the µTitan system, which properties (Checinska Sielaff et al., 2017; Romsdahl et al., 2018,
can extract nucleic acids (and in the future, protein) from a 2019; Singh et al., 2019; Urbaniak et al., 2019). Furthermore,
variety of environmental (manuscript under preparation) and many terrestrial species that have been sent from Earth to
human (Chan et al., 2016a,c) samples now allows for the ability grow on the ISS have become more virulent and antibiotic
to provide end-to-end processing of biological samples from resistant, and have formed more biofilms (Yamaguchi et al.,
raw material to purified nucleic acids, all while in orbit. The 2014), all of which are properties that can affect the health
advantage of the µTitan system over previous ISS studies that of astronauts and the stability of the spacecraft. The µTitan
have used mechanical bead beating or thermal lysing to extract system will also be instrumental for crew monitoring. Having the
nucleic acids is that the µTitan system works for various sample µTitan system on the ISS to process samples and generate data
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Urbaniak et al. µTitan for the ISS
provides the capability to immediately detect and measure several performed experiments in Texas and YNP. JW and NS processed
biomolecules related to the physiological and immunological the raw Nanopore sequencing data and raw Illumina data and
effects of living in space. For example, viral presence during helped design the study. JW, BP, and DS obtained safety protocol
spaceflight is a useful in vivo biomarker of immunodeficiency for using scientific expeditions at YNP with appropriate science
in astronauts (Mehta et al., 2004; Pierson et al., 2005) and permit provided by the YNP and cleared by JPL, Permit #YELL-
was recently demonstrated to positively correlate with immune 2019-SCI-5480. BP and DS participated in sample collections
alterations in astronauts after spaceflight (Mehta et al., 2013). and processing in the YNP and also processing in the MSU
The ability to quantify viral load in astronauts in space, in real lab. RJ carried out the microgravity compatibility study, and FK
time, will provide an efficient means to monitor immune function carried out crew procedure development of the µTitan system.
and to allow for proper countermeasures to be implemented, All authors critically reviewed the manuscript.
such as vaccination strategies, prophylactic antibiotics, or stress
reduction therapies (Kiecolt-Glaser et al., 2010; Kiecolt-Glaser
et al., 2014). FUNDING
This research was supported by the TRISH through Cooperative
CONCLUSION Agreement NNX16AO69A awarded to KV. W. M. Keck
Foundation supported for DS and BP. The NASA postdoctoral
The µTitan instrument is a compact, portable, robust, energy- fellowship supported part of CU time. Preliminary work
efficient device that allows for streamlined and consistent nucleic of this research was supported by a NASA SBIR Contract
acid extractions that requires minimal human labor. It provides (NNX17CP21P) awarded to SW. The funders had no role
the ability to perform complex sample processing on the ISS in the study design, data collection, and interpretation; the
to gain real-time information from environmental and human writing of the manuscript; or the decision to submit the work
samples. While this study has validated the instrument for for publication.
DNA isolation, it has been validated previously for successful
extraction of RNA (Chan et al., 2016b) and thus can be used
to process samples for microbiome, metagenome, transcriptome, ACKNOWLEDGMENTS
and virome analyses on the ISS and possibility on other deep
space missions. The characteristics that make it advantageous Part of the research described in this publication was carried
for space travel also make it suitable for remote settings here out at the Jet Propulsion Laboratory, California Institute of
on Earth. Whether the µTitan system is used on the ISS or on Technology, under a contract with NASA. Researchers associated
Earth, it provides high-quality nucleic acid material for functional with Biotechnology and Planetary Protection Group at JPL are
genomics, microbial monitoring, and detection of biological acknowledged for their facility support.
signatures related to human health and engineering systems.
SUPPLEMENTARY MATERIAL
DATA AVAILABILITY STATEMENT
The Supplementary Material for this article can be found
The datasets presented in this study can be found in online online at: https://www.frontiersin.org/articles/10.3389/fmicb.
repositories. The names of the repository/repositories and 2020.01909/full#supplementary-material
accession number(s) can be found in the article/Supplementary
FIGURE S1 | Summary of sequences detected by shotgun metagenomic
Material. sequencing not represented in the WCMR standard. Heatmap of counts of
non-WCMR-represented species in the 18 samples and 10 controls. These
non-represented species indicate contaminants from sample processing,
AUTHOR CONTRIBUTIONS including extraction reagents, library synthesis, or data analysis artifacts. Each
sample represents the average of sample extraction triplicates.
KV designed the concept and objectives of the study with all FIGURE S2 | Fragment size and molecular weight determination of extracted
authors of the manuscript. CU wrote the manuscript, helped DNA. Agilent Bioanalyzer 2100 data for samples analyzed with the DNA HS chips
design the study, critically analyzed the data, generated all the for Maxwell
TM system extracted samples (laboratory setting in Texas) and µTitan
figures and performed statistical analyses, wrote the R scripts system samples (laboratory setting in Texas and field setting in YNP).
to analyze the NGS data, and processed the WCMR at YNP. FIGURE S3 | Drop tower test. A 2.1 s drop tower test was performed on the
SW invented µTitan and performed all initial studies prior µTitan system and shows the fluid reorientation following a step transition from
to this validation study, and processed the WCMR in Texas 1-g to low-g. The case on the left shows a top-down view of fluid reorientationafter entering low-g. The case on the right shows a well with a probe partially
and in YNP. ST helped develop the WCMR; performed quality submerged. A minor change in fluid orientation occurs after entering low-g, but
control on the WCMR before using it for validation studies; the fluid stays in the preferred position, as desired. Note: Arrows indicate the
and performed NGS, qPCR, Qubit, and bioanalyzer analyses. direction of g prior to entering low-g.
AA performed initial validation of µTitan and processed the TABLE S1 | Table of read counts generated from Illumina HiSeq sequencing. The
WCMR in Texas and YNP. BL, CP, and JW participated and first nine organisms are the ones present in the WCMR.
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Meeting including The New Horizons Forum and Aerospace Exposition (Reston: Conflict of Interest: AA and BL were employed by AI Biosciences, Inc. SW is the
AIAA) co-founder of AI Biosciences, Inc. RJ was employed by IRPI LLC.
Weislogel, M. M., Baker, J. A., and Jenson, R. M. (2011). Quasi-steady capillarity-
driven flows in slender containers with interior edges. J. Fluid Mech. 685, The remaining authors declare that the research was conducted in the absence of
271–305. doi: 10.1017/jfm.2011.314 any commercial or financial relationships that could be construed as a potential
Yamaguchi, N., Roberts, M., Castro, S., Oubre, C., Makimura, K., Leys, N., conflict of interest.
et al. (2014). Microbial monitoring of crewed habitats in space-current status
and future perspectives. Microbes Environ. 29, 250–260. doi: 10.1264/jsme2. Copyright © 2020 Urbaniak, Wong, Tighe, Arumugam, Liu, Parker, Wood, Singh,
me14031 Skorupa, Peyton, Jenson, Karouia, Dragon and Venkateswaran. This is an open-
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