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Article The Development of an Effective Bacterial Single-Cell Lysis Method Suitable for Whole Genome Amplification in Microfluidic Platforms

Yuguang Liu 1, Dirk Schulze-Makuch 2, Jean-Pierre de Vera 3, Charles Cockell 4, Thomas Leya 5 ID , Mickael Baqué 3 and Marina Walther-Antonio 1,6,* 1 Department of Surgery, Division of Surgical Research, Mayo Clinic, Rochester, MN 55905, USA; [email protected] 2 Astrobiology Group, Center of Astronomy and Astrophysics, Technical University, 10623 Berlin, Germany; [email protected] 3 German Aerospace Center (DLR), Institute of Planetary Research, Management and Infrastructure, Astrobiological Laboratories, 12489 Berlin, Germany; [email protected] (J.-P.d.V.); [email protected] (M.B.) 4 School of Physics and Astronomy, University of Edinburgh, Edinburgh EH9 3FD, UK; [email protected] 5 Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Extremophile Research & Biobank CCCryo, 14476 Potsdam, Germany; [email protected] 6 Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, MN 55905, USA * Correspondence: [email protected]; Tel.: +1-507-284-2282



Received: 7 June 2018; Accepted: 19 July 2018; Published: 25 July 2018
 Abstract: Single-cell sequencing is a powerful technology that provides the capability of analyzing a single cell within a population. This technology is mostly coupled with microfluidic systems for controlled cell manipulation and precise fluid handling to shed light on the genomes of a wide range of cells. So far, single-cell sequencing has been focused mostly on human cells due to the ease of lysing the cells for genome amplification. The major challenges that bacterial species pose to genome amplification from single cells include the rigid bacterial cell walls and the need for an effective lysis protocol compatible with microfluidic platforms. In this work, we present a lysis protocol that can be used to extract genomic DNA from both gram-positive and gram-negative species without interfering with the amplification chemistry. Corynebacterium glutamicum was chosen as a typical gram-positive model and Nostoc sp. as a gram-negative model due to major challenges reported in previous studies. Our protocol is based on thermal and chemical lysis. We consider 80% of single-cell replicates that lead to >5 ng DNA after amplification as successful attempts. The protocol was directly applied to Gloeocapsa sp. and the single cells of the eukaryotic Sphaerocystis sp. and achieved a 100% success rate.

Keywords: bacteria lysis protocol; microalgae lysis; single-cell multiple displacement amplification

1. Introduction Phenotypically identical cells from the same population can have dramatic heterogeneity in their behavior. This heterogeneity plays a significant role in various biological processes including tumor progression [1,2] and immune response [3]. Efforts have been focused on exploring the heterogeneous behavior within the same population such as cell growth [4] and drug responses [5–8] using novel molecular reporters and advanced imaging tools. These technologies are effective and popular, however, to better understand the reason behind the different behaviors, it is necessary to identify the variance in the genomes of these genetically similar cells on a single-cell level. Single-cell whole genome sequencing (SC-WGS) is emerging as a promising tool for investigating the genetic diversity and heterogeneity of complex biological systems [9–11]. This technology offers the

Micromachines 2018, 9, 367; doi:10.3390/mi9080367 www.mdpi.com/journal/micromachines Micromachines 2018, 9, 367 2 of 17 ability of identifying genome mutations of a single cell within a population, which is often neglected in standard studies such as metagenomics [12,13]. As a result, SC-WGS is starting to impact our understanding of human physiology and diseases and bring forth new prospects such as evaluating the role of genetic mosaicism [14] in diseases like cancer and facilitating the analyses of uncultured species in environmental studies [15,16]. The key steps in SC-WGS include single-cell isolation, lysis and amplifying genomic DNA from femto to picograms to reach the quantity sufficient for standard library preparation and sequencing (>25 ng) [17,18]. Microfluidic platforms are often used for amplifying genome from single cells due to their unique ability of handling nanoliters of fluid in a controlled manner [19–25], thus, allowing for the isolation of single cells into compartments for lysis and genome amplification [26,27]. Multiple displacement amplification (MDA) [28] has been a popular option for single-cell whole genome amplification (SC-WGA) in microfluidic platforms [29–31]. It is based on ϕ29 DNA polymerase and random primers to replicate template DNA with high fidelity and lower error rates following relatively simple procedures compatible with microfluidic systems [32–34]. So far, most of the SC-WGS applications have been focused on human cells that can be easily lysed using commercial SC-WGA kits. SC-WGS of bacterial species is still rare due to the 1000× lower starting genomic DNA and the rigid and multi-layered cell walls of these microorganisms [35]. The cell wall of gram-positive bacterial species generally consists of a thick layer of peptidoglycan outside of the cytoplasmic cell membrane and, thus, is harder to penetrate. The cell wall of gram-negative bacterial species generally has a thinner peptidoglycan layer between its outer and inner lipid membrane, and are relatively easier to lyse. However, some gram-negative species have much thicker and complex cell walls than most gram-negative microbes and are particularly resilient. For example, the cell wall of cyanobacteria species contains a thick and highly cross-linked peptidoglycan layer and a surface layer composed of polymerized proteins and exopolysaccharide [36], making it especially difficult to penetrate. Many standard bacterium lysis methodologies efficient for bulk studies such as bead beating and high pressure are not applicable to microfluidic platforms [37–39]. Several existing methods effective for the lysis of rigid species such as cyanobacterium were based on mechanical disruptions including sonication and lyophilization [40], but these are low-throughput and not suitable for single-cell studies in microfluidic systems. Others reported genetic-based methods which include expressing a lytic cassette from a bacteriophage in certain strains [41], but these rely on labor-intensive strain-specific genetic engineering and are not generally adaptable. Chemical-based bacterial lysis is a popular option in microfluidic-based SC-WGA. However, the key challenge is to use chemicals with minimal interference with the subsequent MDA amplification chemistry. Due to the challenge of implementing wash steps in the microfluidic-based WGA process, not all chemicals common for effective bacterial cell lysis are applicable. For example, ionic surfactants such as sodium dodecyl sulfate and sarkosyl are often avoided in microfluidic-based WGA because they almost completely inhibit polymerase activities [42]. Chemicals including phenol and spermine have been proven effective for degrading bacterial cell walls, however, due to the toxicity, a fume hood is required, thus, making it difficult to use in microfluidic systems. Freeze-thaw combined with alkaline treatment has been proven highly efficient for the lysis of bacterial single cells for SC-WGA in well plates [43], but requires placing the plates in −80 ◦C freezer for 1 h, therefore it is not applicable for microfluidic experimental setups with complex controls. To overcome these hurdles, others have pursued bacterial SC-WGA in microfluidic chips by pre-lysing the cell population off-chip using ionic surfactants followed by standard in-tube wash prior to introducing them into the chips [42]. This attempt has led to satisfactory results for studying pure cultures cultivated in laboratory settings with an initial on-chip amplification followed by a second round of amplification off-chip in a standard manner. Nevertheless, the off-chip pretreatments are not suitable for bacterial cells in low abundance or are in a complex community such as clinical or environmental samples. Besides, the pre-lyse step would possibly introduce extracellular contaminant Micromachines 2018, 9, 367 3 of 17

DNA when amplifying the extremely low amount of DNA from a single bacterial cell. To perform the entire process on-chip, multiple rounds of amplification can be an alternative to counter the insufficient cell lysis, but it increases the percentage of duplicated reads and thus bias in the data analyses. To extend the use of microfluidic platforms for SC-WGS to microbiological and microbiome research, it is essential to develop protocols suitable for on-chip lysis of a single bacterial cell that circumvents the aforementioned concerns. Moreover, cell wall structures of different bacterial species vary, hence it would be ideal for a protocol to be easily adapted to various species without extensive efforts. Therefore, in this work, we develop a bacterial single-cell lysis protocol as a guideline for MDA-based bacterial SC-WGA in microfluidic platforms that produces >25 ng of genomic DNA per cell, sufficient for downstream library preparation. This on-chip protocol combines three primary bacterial lysis methods which include thermal [15,43,44], enzymatic [45,46] and chemical lysis [47–49] and was tested on both gram-positive and gram-negative bacterial species for subsequent on-chip SC-WGA. In this study, Corynebacterium glutamicum was used as a typical gram-positive model whose cell wall is thicker than most gram-negative species. Besides, Corynebacterium species are increasingly recognized as the occasional causes of prosthetic joint infection associated with significant morbidity [50]; and this disease has a low organism burden and usually involves infection often caused by commensal flora, and thus requiring higher sensitivity and specificity for its identification [51]. Nostoc sp. was chosen as a gram-negative model due to the significant lysis difficulties encountered in previous studies [36,52]. The developed protocol was then tested on Gloeocapsa sp. and Sphaerocystis sp. due to the significant lysis difficulty and the viscous extracellular matrix that largely hinders chemical penetration, and a 100% success was achieved for both species. In addition, Nostoc sp. and Gloeocapsa sp. belong to the cyanobacteria, and cf. Sphaerocystis sp. (hereafter referred to as Sphaerocystis sp.) is a genus of green algae (Chlorophyceae), and these species are of high interest in astrobiological and environmental studies as such taxa were responsible for creating the oxygen atmosphere through photosynthetic activities billions of years ago. We believe that the effective on-chip lysis method that enables successful genome amplification of the chosen species would serve as a guideline for bacterial single-cell genomics in microfluidic platforms, and can be applied to a wide range of applications including biomedical research, environmental studies, and future human space exploration missions.

2. Materials and Methods

2.1. Cell Wall Components of Chosen Bacterial Cells The components of the cell wall are illustrated in Figure1. Generally, the envelope of Corynebacterium spp. consists of an outer membrane primarily composed of polysaccharides and proteins, a cell wall of peptidoglycan layers and a typical plasma membrane bilayer as the inner membrane [53]; while the envelope of cyanobacterial species mainly consists of an external layer composed of exopolysaccharide and polymerized proteins, an outer membrane, a much thicker peptidoglycan layer and an inner cytoplasmic membrane [54]. Sphaerocystis sp. is a genus of green algae (Chlorophyta), in which the cell wall surrounds the cytoplasm membrane and usually is composed of microfibrillar polysaccharides and is covered by an extracellular polysaccharide matrix [55,56]. Therefore, the lysis protocol was designed to sequentially break through the cell envelope from the outermost to the innermost layer with minimal interference with ϕ29 DNA polymerase activity. Micromachines 2018, 9, 367 4 of 17 Micromachines 2018, 9, x FOR PEER REVIEW 4 of 17

Figure 1.1. AA representative representative illustration illustration of theof cellthe envelopscell envelops of Corynebacterium, of Corynebacterium, cyanobacterium cyanobacterium species, species,and green and algae. green algae.

Others have proven that following the lysis instruction of the Relpli-g Single Cell kit (Qiagen) Others have proven that following the lysis instruction of the Relpli-g Single Cell kit (Qiagen) would achieve a 90% amplification rate of single Escherichia coli [27]. However, only 30% of C. would achieve a 90% amplification rate of single Escherichia coli [27]. However, only 30% of glutamicum single cells were amplified with an average of 15.78 ng DNA in the same manner. This is C. glutamicum single cells were amplified with an average of 15.78 ng DNA in the same manner. likely due to the fact that the peptidoglycan layer of gram-positive species is multilayered, with a This is likely due to the fact that the peptidoglycan layer of gram-positive species is multilayered, with thickness range of 30–100 nm, while the gram-negative species has a single-layered peptidoglycan a thickness range of 30–100 nm, while the gram-negative species has a single-layered peptidoglycan layer of 2–10 nm [54,57]. This shows that additional treatments are necessary to sufficiently lyse layer of 2–10 nm [54,57]. This shows that additional treatments are necessary to sufficiently lyse species species with thicker cell walls. with thicker cell walls.

2.2. Cell Preparation C. glutamicum (donated byby Dr.Dr. RobinRobin Patel,Patel, MayoMayo Clinic,Clinic, Rochester, Rochester, MN, MN, USA) USA) was was cultured cultured in in a ° anutrient nutrient broth broth (DB) (DB) at at 37 37◦ CC and and harvested harvested duringduring loglog phasephase andand diluteddiluted in a sample diluent (0.08% 6 Pluronic F127 (Sigma Aldrich, St.St. Louis,Louis, MO,MO, USA)USA) inin PhosphatePhosphate BufferBuffer SalineSaline (PBS))(PBS)) to to ~10 ~106/mL/mL to facilitate single-cell trapping. The The Antarctic Antarctic strain strain CCCryo CCCryo 231-06 231-06 of of the the cyanobacterium cyanobacterium NostocNostoc spsp.. and the Arctic Arctic strain strain CCCryo CCCryo 101-99 101-99 of of cf. cf. SphaerocystisSphaerocystis spsp.. (cf. (cf. = Latin.: = Latin.: confer, confer, meaning meaning “needs “needs to be to discussed”;be discussed”; the the taxonomic taxonomic identity identity of ofthis this strain strain is isnot not yet yet fully fully resolved) resolved) were were obtained obtained from from the Culture Collection of Cryophilic Algae (CCCryo) at th thee Branch Bioanalytics and Bioprocesses of the Fraunhofer Institute for Cell Therapy and Immunology (IZI-BB) in Potsdam. They They were were collected, cultured, and maintained in cooperation with the German Aerospace Center (DLR) Berlin. Gloeocapsa spsp.. was was obtained obtained from from the University of Edinburgh, Edinburgh, UK. All samples were received in the desiccated form and re-suspended in the sample diluent.

2.3. Microfluidic Microfluidic Experimental Setup The study was performed in an optofluidicoptofluidic platform initially developed by Landry et al. [27] [27] and reconstructed at the Mayo Clinic (Rochester (Rochester,, MN, USA). Briefly, Briefly, this platform integrates a microscope (Nikon Eclipse, Melville, NY, NY, USA), optical tweezers (1064 mn, Thorlabs, Newton, NJ, NJ, USA), and a customized Polydimethylsiloxane (PDMS) microfluidic microfluidic chip with 12 parallel reaction systems (Figure 22a).a). EachEach reactionreaction systemsystem hashas aa seriesseries ofof valvesvalves thatthat controlcontrol thethe openingopening andand closingclosing of the the chambers, chambers, allowing allowing for for the the on-demand on-demand creati creationon of isolated of isolated microenvir microenvironmentsonments (Figure (Figure 2b). 2Theb). detailsThe details of the of thedevice device fabrication fabrication protocol protocol are are provided provided in in the the Supplementary Supplementary Materials. Other microfluidicmicrofluidic systems such as droplet microfluidics microfluidics [58–60] [58–60] are attractive alternatives due to their high-throughput, however, these are based-on randomrandom encapsulation based on probability which is more suitable for the studies of pure cultures. Due Due to to the the complexity complexity of of the the NostocNostoc sp.,sp., GloeocapsaGloeocapsa spsp.. and Sphaerocystis spsp.. we we obtained, optical tweezers were were chosen to ensure higher target single-cell confidenceconfidence with minimal contaminants. In thisthis study,study, the the microfluidic microfluidic channels channels for samplefor sample introduction introduction were were pre-soaked pre-soaked in the chipin the diluent chip diluent(0.04% Pluronic(0.04% Pluronic F127 in F127 PBS) in for PBS) 30 min for 30 prior min to prio experimentsr to experiments to prevent to prevent the cells the from cells sticking from sticking to the toPDMS the PDMS channel channel surface surface during during the cell the sorting. cell sortin Samplesg. Samples can be can introduced be introduced into the into chip, the and chip, single and singlecells can cells be can trapped be trapped and transported and transported into microchambers into microchambers by optical by traps optical (Figure traps2 c).(Figure The valves 2c). The of valvesthese chambers of these thenchambers can be closedthen can to isolate be closed the single to isolate cells. Visuallythe single identifiable cells. Visually contaminating identifiable cells contaminating cells can be trapped and transported out of the chambers to ensure only the target cell is in the chamber prior to the lysis step. The laser power at the objective was measured to be 50 mW,

Micromachines 2018, 9, 367 5 of 17

Micromachinescan be trapped 2018, 9 and, x FOR transported PEER REVIEW out of the chambers to ensure only the target cell is in the chamber5 of 17 prior to the lysis step. The laser power at the objective was measured to be 50 mW, which was proven whichby others was with proven marginal by others effects with on cell marginal viability effects [61,62 on]. Genomic cell viability mutation [61,62]. of bacterialGenomic cells mutation under theof bacterialeffect of lasercells powerunder hasthe noteffect been of reportedlaser power to the has best not of been our knowledge, reported to however, the best itof would our knowledge, be valuable however,to characterize it would this be aspect valuable in our to futurecharacterize single-cell this aspect genomic in our studies. future Reagents single-cell include genomic lysis studies. buffers Reagentsand DNA include polymerase lysis buffers can be sequentiallyand DNA polymerase added to thecan isolated be sequentially cells to perform added to chemical the isolated reactions. cells toThe perform amplified chemical product reactions. can be collected The amplified from the prod outletuct ports can be of thecollected chip and from transferred the outlet into ports microwell of the chipplates and for transferred downstream into processing. microwell plates All the for supplies downstream and reagents processing. were All filtered the supplies (0.2 µm), and autoclaved reagents wereor UV-sterilized, filtered (0.2 exceptµm), autoclaved for the DNA or UV-sterilized, polymerase. Ten except single-cell for the reactionsDNA polymerase. and two negative Ten single-cell control reactionsreactions and were two performed negative in control each test. reactions were performed in each test.

FigureFigure 2. 2. AnAn overview overview of of the the optofluidic optofluidic single-cell single-cell whole whole genome genome amplification amplification (SC-WGA) (SC-WGA) platform. platform. ((aa)) This This platform platform integrates integrates an an advanced advanced microscope microscope,, optical optical tweezers, tweezers, and anda microfluidic a microfluidic device. device. The deviceThe device consists consists of 12 of parallel 12 parallel reaction reaction systems. systems. (b) The (b) Thedouble-layer double-layer microfluidic microfluidic device device consists consists of flowof flow channels channels in the in the top top layer layer and and control control channe channelsls inin the the bottom bottom layer. layer. Chambers Chambers are are formed formed by by pressuringpressuring control control channels channels into into flow flow channels channels at at their their junctions. junctions. ( (cc)) Time-elapsed Time-elapsed images images of of a a single single NostocNostoc sp.sp. cell cell is is trapped trapped and and moved moved into into a chamber. a chamber. The The inset inset illustrates illustrates the thelocation location of the of cell the in cell the in channel.the channel.

2.4.2.4. Choice Choice of of Lysis Lysis Buffer Buffer Components Components TheThe study study started started with with REPLI-g REPLI-g Single Single Cell Cell Kit Kit (Qiagen, (Qiagen, Germantown, Germantown, MD, MD, USA) USA) instruction instruction as theas thebaseline, baseline, along along with with the propriety the propriety D2 lysis D2 lysisbuffer buffer (concentrated (concentrated alkaline) alkaline) supplied supplied in the in kit. the Due kit. toDue the toinsufficiency the insufficiency of the standard of the standard alkaline-based alkaline-based lysis protocol, lysis protocol, we hypothesized we hypothesized that the addition that the ofaddition brief heat-shock of brief heat-shock steps, an steps, appropriate an appropriate amount amount of lysozyme of lysozyme (Epicenter, (Epicenter, Madison, Madison, WI, WI, USA), USA), DithiothreitolDithiothreitol (DTT)(DTT) (Biorad,(Biorad, Hercules, Hercules, CA, CA, USA) USA) and and Ethylenediaminetetraacetic Ethylenediaminetetraacetic acid (EDTA)acid (EDTA) would wouldsignificantly significantly improve improve the lysis the success lysis success for hard-to-lyse for hard-to-lyse species species according according to the known to the known effects ofeffects each ofof each these of chemicals these chemicals on the bacterial on the cellbacterial wall [42cell,46 wall,63]. [42,46,63]. Note that EDTANote that was EDTA reported was for reported enabling for the enablingpermeability the ofpermeability the outer membrane of the outer by chelating membrane divalent by chelating cations thatdivalent stabilizes cations negatively-charged that stabilizes negatively-charged sugars but with minor effects [36], therefore, we kept EDTA concentration at a constant value of 0.5 mM without further optimization. Details of the concentration of lysozyme and DTT used in the lysis buffer during the optimization process are presented in the Results and Discussion section.

Micromachines 2018, 9, 367 6 of 17 sugars but with minor effects [36], therefore, we kept EDTA concentration at a constant value of 0.5 mM without further optimization. Details of the concentration of lysozyme and DTT used in the lysis buffer during the optimization process are presented in the Results and Discussion section.

2.5. Microfluidic Bacterial Lysis for SC-WGA Workflow The general workflow is shown in Figure3. After single-cell isolation in the microfluidic chip, a heat-shock was performed by alternately placing the microfluidic chip on a hotplate and a cold block Micromachines 2018, 9, x FOR PEER REVIEW 6 of 17 for a controlled amount of time. Lysis reagents were introduced sequentially and the microfluidic chip was placed2.5. Microfluidic on a hotplate Bacterial Lysis for reagentfor SC-WGA incubation. Workflow After lytic reactions, the neutralized buffer in the kit was addedThe general to terminate workflow DNA is shown denaturation in Figure 3. After at room single-cell temperature. isolation in the The microfluidic polymerase chip, was a mixed according toheat-shock the kit’s was instruction performed by and alternately added placing into the the reaction microfluidic chambers, chip on a hotplate and the and chip a cold was block placed on a hotplate atfor 32 a◦ Ccontrolled for 16 h.amount The amplificationof time. Lysis reagents reaction were was introduced terminated sequentially by incubating and the microfluidic the microfluidic chip was placed on a hotplate for reagent incubation. After lytic reactions, the neutralized buffer in chip at 65 ◦C for 3 min and cooled on ice. Gel-loading pipette tips were inserted into the outlet ports of the kit was added to terminate DNA denaturation at room temperature. The polymerase was mixed the chip, andaccording nuclease-free to the kit’s water instruction was and introduced added into into the reaction the chip chambers, to flush and the the amplified chip was placed product on into the ◦ pipette tipsa untilhotplate the at fluid32 °C for level 16 h. reached The amplification the 20 µ reactionL mark. was The terminated product by was incubating collected the microfluidic and stored at 4 C, and a high-sensitivitychip at 65 °C for Qubit 3 min assay and cooled (Thermo on ice. Fisher,Gel-loading Waltham, pipette tips MA, were USA) inserted was into performed the outlet ports to assess the of the chip, and nuclease-free water was introduced into the chip to flush the amplified product into amount of the amplified genomic DNA from single cells. If the amplified genomic DNA from a single the pipette tips until the fluid level reached the 20 µL mark. The product was collected and stored at cell was >254 °C, ng and while a high-sensitivity the DNA is Qubit not detectable assay (Therm ino negativeFisher, Waltham, controls, MA, weUSA) considered was performed it a to successful lysis and amplification.assess the amount Noof the evaporation amplified genomic was DNA observed from single during cells. the If the process amplified at genomic elevated DNA incubation temperaturesfrom (up a single to 95 cell◦C) was as >25 the ngfluid while wasthe DNA contained is not detectable in closed in negative chambers. controls, Details we considered of the lysis it buffer a successful lysis and amplification. No evaporation was observed during the process at elevated incubation time and temperature used during the protocol optimization process are presented in the incubation temperatures (up to 95 °C) as the fluid was contained in closed chambers. Details of the Results andlysis Discussion buffer incubation section. time Note and that temperature the results used from during the the protocol protocol development optimization process processes are that did not lead topresented expected in resultsthe Results (>25 and ng)Discussion were section. also presented; Note that the however, results from we the continued protocol development the optimization until the averageprocesses amount that did ofnot DNAlead to fromexpected a single results cell(>25 wasng) were amplified also presented; to >25 however, ng. we continued the optimization until the average amount of DNA from a single cell was amplified to >25 ng.

Figure 3. An overview of the workflow of single-cell isolation, lysis, and DNA amplification in a Figure 3. Anmicrofluidic overview chip. ofThe the blue workflow rectangles represent of single-cell standard isolation, steps in an on-chip lysis, andSC-WGA DNA process. amplification The in a microfluidicgreen chip. box highlights The blue the genera rectanglesl steps of represent the lysis protocol standard developed steps and in optimized. an on-chip SC-WGA process. The green box highlights the general steps of the lysis protocol developed and optimized.

Micromachines 2018, 9, x FOR PEER REVIEW 7 of 17 Micromachines 2018, 9, 367 7 of 17 3. Results and Discussion 3. Results and Discussion 3.1. Optimization of Heat-Shock Treatment on C. glutamicum 3.1. OptimizationHeat-shock is of a Heat-Shock physical Treatmenttreatment oncommonly C. glutamicum used in bacterial transformation and lysis as it altersHeat-shock the fluidity is of a physicalthe cell membrane treatment commonly and creates used pores in bacterialdue to the transformation sudden change and in lysis temperature as it alters [63–66].the fluidity We oftested the cell the membrane effects of heat-shock and creates temperature pores due to range the sudden on C. changeglutamicum in temperature as a pretreatment [63–66]. priorWe tested to following the effects the of heat-shockstandard temperatureinstruction range(Figure on 4).C. glutamicumPerforming asheat-shock a pretreatment at different prior to temperaturesfollowing the standardresulted instructionin different (Figure single-cell4). Performing amplification heat-shock rates at and different yields. temperatures Ice/65 °C resultedslightly increasedin different the single-cell single-cell amplification amplification ratesrate by and 10% yields. and doubled Ice/65 the◦C averag slightlye amplified increased DNA the single-cell amount, butamplification the difference rate in by amplified 10% and DNA doubled amount the between average th amplifiede two conditions DNA amount, is not statistically but the difference significant. in However,amplified DNAthe wide-spread amount between amplified the twoDNA conditions amount is(0.77–57.2 not statistically ng) suggests significant. that the However, ice/65 the°C treatmentwide-spread was amplified sufficient DNA for certain amount single-cell (0.77–57.2 replicates, ng) suggests but that hadthe a minor ice/65 effect◦C treatment on the others was sufficientpossibly duefor certainto the single-cellstochastic replicates,cell wall features but had aof minor indivi effectdual oncells. the To others test possiblyif the heat-shock due to the with stochastic a higher cell intensitywall features would of individualhave a distinct cells. effect To test on if thethe heat-shockamplification with rate a higher for C. intensityglutamicum would single have cells, a distinct more aggressiveeffect on the heat-shock amplification tests ratewere for attempted.C. glutamicum Whensingle the upper cells, temperature more aggressive was increased heat-shock to tests 90 °C, were no detectableattempted. DNA When was the obtained upper temperature after amplification. was increased We assume to 90 ◦C, that no detectablethe amplification DNA was failure obtained was causedafter amplification. by the over-denaturation We assume that of DNA the amplification rather than DNA failure degradation was caused at by high the temperature, over-denaturation as DNA of degradationDNA rather thanstarts DNA at 100 degradation °C [67] and at highgenerally temperature, its melting as DNAtemperature degradation lies between starts at 10050–100◦C[ 67°C] dependingand generally on itsfactors melting including temperature genome lies size between and gu 50–100anine-cytosine◦C depending content on factors[68]. Since including the D2 genome buffer alreadysize and contained guanine-cytosine DNA denaturing content [68 ].agents, Since theincubati D2 bufferng it already at elevated contained temperature DNA denaturing could possibly agents, accelerateincubating DNA it at elevateddenaturation. temperature could possibly accelerate DNA denaturation.

FigureFigure 4. TheThe temperature temperature range range effect effect of of heat-shock on on the C. glutamicum lysislysis for for SC-WGA. SC-WGA. After After heat-shockheat-shock treatment, treatment, the the proprietary proprietary D2 D2 alkaline alkaline ly lysissis buffer buffer was was added and incubated at at 65 °C◦C for 10 min, according to the manufacturer’smanufacturer’s instruction. NN == 10 10 single single cells cells per per condition, condition, error error bars representrepresent standard deviation.

WhenWhen the lower temperature was decreased toto −−8080 °C,◦C, only only 10% 10% of of the the single single cells cells amplified amplified and and resultedresulted in 2.9 ng DNA,DNA, whilewhile −−2020 °C/65◦C/65 °C◦C led led to to 90% 90% of of single-cell single-cell amplification amplification with with 6.81 6.81 ng ng of DNA.DNA. Even Even though though a a more aggressive heat-shock is is expected to exert stronger destabilization effect effect onon the bacterial cell wall through mechanical fracturing, DNA shearing could happen during ice crystalcrystal formation formation [69] [69] at at extremely extremely low low temperatures temperatures which which eventually eventually lead lead to to amplification amplification failure. failure. We further investigated the effect of the number of heat-shock cycles by repeating repeating the the test test with with three three cyclescycles of ice/65 ice/65 °C◦C treatment. treatment. This This raised raised the the lysi lysiss rate rate by by 10% 10% but but lowered lowered the the amplified amplified DNA DNA to to only only aa few nanograms,nanograms, which which suggests suggests that that the the template template DNA DNA quality quality may havemay beenhave compromisedbeen compromised during duringthe extended the extended heat-shock heat-shock cycles forcyclesC. glutamicum for C. glutamicum. .

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3.2. Optimization of Lysozyme Treatment on C. glutamicum 3.2. Optimization of Lysozyme Treatment on C. glutamicum To further increase the lysis rate and amplification yield, lysozyme was used after the heat-shock due toTo its further well-known increase effect the lysis of ratedigesting and amplification the peptidoglycan yield, lysozyme layer [46,63]. was usedWe continued after the heat-shock the lysis optimizationdue to its well-known based on effectthe optimal of digesting lysis condition the peptidoglycan achieved so layer far [with46,63 ].other We variables continued unaltered the lysis (Figureoptimization 5). After based one oncycle the of optimal ice/65 °C lysis heat-shock, condition 100 achieved U/µL Ready-Lyse so far with lysozyme other variables was added unaltered and incubated(Figure5). at After 37 °C one for cycle10 min; of after ice/65 that,◦C the heat-shock, D2 buffer was 100 U/addedµL Ready-Lyseand incubated lysozyme at 65 °C wasfor 10 added min priorand incubated to stopping at the 37 lytic◦C for reaction. 10 min; Surprisingly, after that, the no D2 amplification buffer was was added measured and incubated in any of at the 65 single-◦C for cell10 min replicates. prior to As stopping a common the lytic bacterial reaction. DNA Surprisingly, extraction reagent, no amplification lysozyme was has measured not been inreported any of theto causesingle-cell DNA replicates. degradation As a [46]; common besides, bacterial 200 U/µL DNA of extraction lysozyme reagent, poses lysozymemarginal hasdeleterious not been effect reported on MDA-basedto cause DNA genome degradation amplification [46]; besides, and has 200 been U/µ widelyL of lysozyme used in various poses marginal MDA reactions deleterious [42,58]. effect Due on toMDA-based these reasons, genome we assume amplification that the and released has been DN widelyA was used overly in various denatured MDA during reactions the [incubation42,58]. Due of to D2these at 65 reasons, °C. We we verified assume this that assumption the released by DNA lowering was overlythe D2 denatured buffer incubation during the temperature incubation to of 37 D2 °C, at and65 ◦ C.this We led verified to 10% this of the assumption amplification by lowering of single the C. D2glutamicum buffer incubation cells with temperature 1.76 ng DNA. to 37However,◦C, and thisthe stillled topoor 10% performance of the amplification suggests that of single the combinedC. glutamicum use of heat-shock,cells with 1.76 lysozyme, ng DNA. and However, alkaline reagents the still waspoor too performance aggressive suggests for C. thatglutamicum the combined cells because use of heat-shock, the results lysozyme, above showed and alkaline that the reagents ice/65 was°C treatmenttoo aggressive combined for C. glutamicumwith the alkaline-basedcells because the lysis results was above already showed sufficient that thefor ice/65 certain◦C single-cell treatment replicates.combined withTo find the alkaline-basedout an optimal lysis condition, was already we used sufficient only forlysozyme certain single-celland the D2 replicates. buffer at To37 find °C withoutout an optimal heat-shock condition, treatment, we and used this only resulted lysozyme in 20% and of the the D2 single-cell buffer at genome 37 ◦C without amplification heat-shock with antreatment, average andof 11.72 this resultedng DNA, in and 20% the of thesingle-cell single-cell amplification genome amplification rate further with increased an average to 30% of 11.72with an ng averageDNA, and of 9.5 the ng single-cell of DNA when amplification the D2 buffer rate further was incubated increased atto room 30% temperature, with an average thus ofconfirming 9.5 ng of ourDNA assumption. when the D2 These buffer results was incubatedsuggest that at room the elevated temperature, incubation thus confirming temperature our for assumption. the D2 alkaline These bufferresults might suggest have that lysed the elevatedthe C. glutamicum incubation cells temperature more effectively for the but D2 may alkaline have buffer caused might damages have to lysed the templatethe C. glutamicum DNA; moreover,cells more this effectively would butnarrow may the have window caused damagesfor finding to theout template an ideal DNA;lysis condition moreover, whilethis would maintaining narrow thean windowacceptable for findingDNA integrity out an ideal for lysissuccessful condition amplification. while maintaining For this an reason, acceptable we decidedDNA integrity to proceed for successful with the amplification.protocol optimization For this reason, with the we decidedincubating to proceedD2 lysis with buffer the at protocol room temperatureoptimization rather with thethan incubating 37 °C since D2 the lysis results buffer from at room these temperature two conditions rather were than not 37 ◦statisticallyC since the different.results from these two conditions were not statistically different.

FigureFigure 5. 5. TheThe optimization optimization of of the the combined combined effect effect of ofthermal thermal treatment, treatment, lysozyme, lysozyme, and and DTT DTT on onC. glutamicumC. glutamicum lysislysis to toreach reach the the optimal optimal co conditionndition for for subsequent subsequent SC-WGA. SC-WGA. NN == 10 10 single single cells cells per per condition,condition, error error bars repr representesent standard deviation.

3.3.3.3. Optimization Optimization of of Lysozy Lysozymeme Combined with DTT Treatment on C.C. glutamicum AlthoughAlthough using using Ready-Lyse Ready-Lyse lysozyme lysozyme followed followed by by the the D2 D2 buffer buffer did did not not lead to desirable improvementsimprovements in in single-cell single-cell amplification, amplification, we we assume assume that that the the lysozyme lysozyme failed failed to to reach reach and and digest digest thethe peptidoglycan peptidoglycan layers layers in in an an effective effective manner. manner. Therefore, Therefore, the the sulfhydryl sulfhydryl compound compound DTT DTT was was used used

Micromachines 2018, 9, 367 9 of 17 simultaneously with lysozyme because it is reported as an agent for degrading the external layers due to its ability of breaking disulfide bonds, disrupting polysaccharide, and altering the extracellular matrix of biofilms [70–72]. We used 100 mM DTT and 100 U/µL lysozyme simultaneously and incubated them at 37 ◦C for 10 min, followed by adding the D2 buffer and incubating it at room temperature for 10 min. Expectedly, the C. glutamicum single-cell amplification rate was raised to 80% with an average of 14.75 ng of DNA. However, under this lysis condition, most of the single-cell replicates amplifiedMicromachines to 2018 only, 9, x FOR 6–17 PEERng, REVIEW and merely one single cell amplified to 48 ng,9 thusof 17 leading to the widespread standard deviation. This shows that the same lysis condition could lead to significant differences insimultaneously cell lysis due with to lysozyme the stochastic because itfeatures is reported of as differentan agent for single degrading cells the within external thelayers same species, due to its ability of breaking disulfide bonds, disrupting polysaccharide, and altering the extracellular which explainsmatrix why of itbiofilms is generally [70–72]. easierWe used to 100 extract mM DTT DNA and from 100 U/µL bulk lysozyme cells without simultaneously the need and for intensive lysis protocolincubated development. them at 37 Comparing°C for 10 min, withfollowed previous by adding conditions the D2 buffer inand Figure incubating5, the it at improvement room of single-cell lysistemperature and amplification for 10 min. Expectedly, by adding the C. glutamicum DTT implies single-cell that amplification DTT assisted rate was the raised bacterial to 80% cell lysis by with an average of 14.75 ng of DNA. However, under this lysis condition, most of the single-cell permeating thereplicates external amplified layers to only to enable6–17 ng, and lysozyme merely one to single degrade cell amplified peptidoglycan to 48 ng, thus layers. leading As to an effort to further improvethe widespread the amplification, standard deviation. the DTT This concentration shows that the same was lysis increased condition could to 200 lead mM to significant while the lysozyme remained 100differences U/µL, however, in cell lysis due no to significant the stochastic improvement features of different in thesingle amplification cells within the same rate species, was achieved but which explains why it is generally easier to extract DNA from bulk cells without the need for the DNA amountintensive was lysis increased protocol development. by 21%, with Comparing the highest with andprevious lowest conditions amplified in Figure DNA 5, the amount being 64 ng and 6 ngimprovement respectively. of single-cell These lysis results and amplificatio imply thatn by 200 adding mM DTT DTT implies is sufficient that DTT assisted to break the through the bacterial cell lysis by permeating the external layers to enable lysozyme to degrade peptidoglycan external layer for lysozyme to take effect. Further improvement of cell wall penetration would possibly layers. As an effort to further improve the amplification, the DTT concentration was increased to 200 rely on the increasemM while of lysozymethe lysozyme concentration. remained 100 U/µL, however, no significant improvement in the To find outamplification whether rate DTT was orachieved lysozyme but the was DNA more amount significant was increased to by the 21%, lysis with process, the highest 100 and mM DTT and lowest amplified DNA amount being 64 ng and 6 ng respectively. These results imply that 200 mM 200 U/µL lysozyme was used instead, and this achieved a 100% single C. glutamicum amplification, DTT is sufficient to break through the external layer for lysozyme to take effect. Further improvement however, theof average cell wall penetration DNA was would lowered possibly by rely 30%. on the These increase results of lysozyme imply concentration. that the increased concentration of lysozyme penetratedTo find out the whether outer DTT membrane or lysozyme was of allmore the significantC. glutamicum to the lysis process,replicates 100 mM in theDTT presence of and 200 U/µL lysozyme was used instead, and this achieved a 100% single C. glutamicum 100 mM DTT but with rather low speed. This can explain why only 2 replicates were amplified to amplification, however, the average DNA was lowered by 30%. These results imply that the increased >25 ng of DNAconcentration while the of rest lysozyme remained penetrated 6–10 the ng. outer Thus, membrane both of DTT all the and C. lysozymeglutamicum replicates played in a the significant role and the decreasedpresence concentration of 100 mM DTT but of eitherwith rather would low spee compromised. This can explain the amplification. why only 2 replicates Therefore, were as a final amplified to >25 ng of DNAµ while the rest remained 6–10 ng. Thus, both DTT and lysozyme played attempt, 200 mMa significant DTT and role 200and the U/ decreasedL lysozyme concentration was used of either at the would same compromise time; as the predicted, amplification. this achieved a 100% amplificationTherefore, rate as a with final anattempt, average 200 mM of DTT 29 ng and DNA; 200 U/µL and lysozyme among was the used amplified at the same single-cell time; as replicates, 90% led to >25predicted, ng DNA, this improvingachieved a 100% significantly amplification comparedrate with an withaverage the of previous29 ng DNA; attemptand among (p the-value = 0.017). amplified single-cell replicates, 90% led to >25 ng DNA, improving significantly compared with the C. glutamicum After the expectedprevious success attempt of(p-value lysis = and 0.017). amplification After the expected of singlesuccess of lysis and amplificationwas achieved, of single C. we assessed the integrity ofglutamicum the amplified was achieved, DNA we from assessed single the integrityC. glutamicum of the amplifiedcells DNA in Tapestation from single C. glutamicum (Agilent 2200, D1000 Screentape, Santacells in Clara, Tapestation CA, (Agilent USA), 2200, and D1000 the results Screentape, show Santa that Clara, the CA, DNA USA), was and notthe results degraded show (Figure6). that the DNA was not degraded (Figure 6).

Figure 6. The tape station results of amplified DNA from single C. glutamicum cells using the Figure 6. The tape station results of amplified DNA from single C. glutamicum cells using the optimized protocol. The DNA was not degraded. The results from three single C. glutamicum cells were shown, with one negative control. Micromachines 2018, 9, x FOR PEER REVIEW 10 of 17

optimized protocol. The DNA was not degraded. The results from three single C. glutamicum cells were shown, with one negative control. Micromachines 2018, 9, 367 10 of 17 3.4. The Cyanobacteria Species’ Cell Wall Description

3.4. TheAs Cyanobacteriaa gram-negative Species’ species, Cell cyanobacteria Wall Description cell wall structure is perhaps the largest and the most diverse group of bacteria and is among the most challenging to break through. Some cyanobacteria can haveAs a gram-negativepeptidoglycan species,layers of cyanobacteria over 700 nm [73], cell walland structurethe degree is of perhaps cross-linking the largest of peptidoglycan and the most diverselayer is group20–33% of higher bacteria than and most is among of the gram-negativ the most challenginge species, to similar break to through. that of gram-positive Some cyanobacteria species can(56–63%) have peptidoglycan[74]. In addition, layers cyanobacterial of over 700 nm oute [73r ],membranes and the degree components of cross-linking include of carotenoids peptidoglycan and layer is 20–33% higher than most of the gram-negative species, similar to that of gram-positive species unusual fatty acids that are linked to the peptidoglycan layers via bridge-like coiled-coil domains [54]. (56–63%)These features [74]. Inmake addition, cyanobacterium cyanobacterial perhaps outer the membranes most difficult components microbes includeto lyse for carotenoids amplification and unusualand thus fatty has acids been that rarely are linkedstudied to on the a peptidoglycan single-cell level. layers Therefore, via bridge-like it is necessary coiled-coil to domains rely on [ 54the]. Thesecombined features effects make of cyanobacterium heat-shock and perhaps aforemention the mosted difficult reagents microbes with much-increased to lyse for amplification intensity and to thusachieve has the been goal. rarely studied on a single-cell level. Therefore, it is necessary to rely on the combined effects of heat-shock and aforementioned reagents with much-increased intensity to achieve the goal. 3.5. The Optimization of the Heat-Shock Treatment on Nostoc sp. 3.5. The Optimization of the Heat-Shock Treatment on Nostoc sp. We started the investigation by treating the single cells with 200 U/µL Ready-Lyse lysozyme We started the investigation by treating the single cells with 200 U/µL Ready-Lyse lysozyme after after heat-shock (Figure 7). Unlike C. glutamicum, a cycle of ice/65 °C heat-shock did not pose any heat-shock (Figure7). Unlike C. glutamicum, a cycle of ice/65 ◦C heat-shock did not pose any detectable detectable effect on Nostoc sp., instead, a cycle of −20 °C/65 °C heat-shock led to 10% of single-cell effect on Nostoc sp., instead, a cycle of −20 ◦C/65 ◦C heat-shock led to 10% of single-cell amplification amplification with 2.38 ng of DNA. However, no amplification was observed when a cycle of −80 with 2.38 ng of DNA. However, no amplification was observed when a cycle of −80 ◦C/65 ◦C °C/65 °C heat-shock was performed. These phenomena show that bacteria with thicker walls are able heat-shock was performed. These phenomena show that bacteria with thicker walls are able of of enduring more aggressive thermal treatment with a larger temperature difference. Even so, enduring more aggressive thermal treatment with a larger temperature difference. Even so, extreme extreme temperatures are not ideal treatments for cell lysis even for species with thick, rigid and temperatures are not ideal treatments for cell lysis even for species with thick, rigid and multilayered multilayered cell walls due to the possibility of DNA shearing during ice crystal formation although cell walls due to the possibility of DNA shearing during ice crystal formation although the cell walls the cell walls may be shattered to some extent. To reinforce the Nostoc sp. cell wall destabilization may be shattered to some extent. To reinforce the Nostoc sp. cell wall destabilization while preserving while preserving the integrity of the DNA template, a −20 °C/65 °C heat-shock was performed for 3 the integrity of the DNA template, a −20 ◦C/65 ◦C heat-shock was performed for 3 cycles consecutively. cycles consecutively. As expected, the single-cell amplification rate was increased to 50% but the As expected, the single-cell amplification rate was increased to 50% but the average DNA amount average DNA amount still remained low. The further increase in the number of cycles did not induce still remained low. The further increase in the number of cycles did not induce statistically significant statistically significant improvement on either the amplification rate or the DNA amount. The tests improvement on either the amplification rate or the DNA amount. The tests on both C. glutamicum on both C. glutamicum and Nostoc sp. cells show that appropriate temperatures and cycles of heat- and Nostoc sp. cells show that appropriate temperatures and cycles of heat-shock apparently assisted shock apparently assisted the cell wall destabilization but only to a limited extent. the cell wall destabilization but only to a limited extent.

FigureFigure 7.7. TheThe temperaturetemperature rangerange effecteffect andand cyclescycles ofof heat-shockheat-shock ononNostoc Nostocsp. sp. lysislysis forfor SC-WGA.SC-WGA. AfterAfter heat-shockheat-shock treatment,treatment, a a mixturemixture of of 200200 U/U/µLµL Ready-LyseReady-Lyse lysozymelysozyme andand 0.50.5 mMmM EDTAEDTA waswas addedadded andand incubatedincubated atat 3737 ◦°CC forfor 22 h.h. The D2 alkaline lysis bufferbuffer waswas thenthen addedadded andand incubatedincubated atat 6565 ◦°CC for 2 h. NN == 1010 singlesingle cellscells perper condition;condition; errorerror barsbars representrepresent standardstandard deviation.deviation.

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3.6. The Optimization of Lysozyme Combined with the DTT Treatment on Nostoc sp. 3.6. TheTherefore, Optimization chemical of Lysozyme effects Combined were investigat with the DTTed to Treatment sequentially on Nostoc degrade sp. the multi-layered cyanobacterialTherefore, cell chemical walls following effects werethe heat-shock investigated treatment. to sequentially Even though degrade lysozyme the multi-layeredis known for degradingcyanobacterial peptidoglycan cell walls followinglayers [75,76] the and heat-shock interfering treatment. with nucleic Even acid though polymerase lysozyme [42,77], is known for the for cyanobacterium,degrading peptidoglycan the peptidoglycan layers [75 ,76layers] and are interfering encapsulated with nucleicby the acidthick polymerase external layer [42, 77and], forouter the membranecyanobacterium, that prevents the peptidoglycan lysozyme to layers permeate are encapsulatedthrough. Therefore, by the DTT thick was external used layeras an andagent outer for breakingmembrane disulfide that prevents bonds and lysozyme disrupt to polysa permeateccharide through. of the Therefore, external layer DTT [70–72]. was used as an agent for breakingBased disulfide on the optimal bonds and lysis disrupt condition polysaccharide investigated of to the this external point, different layer [70 –concentrations72]. of DTT and itsBased incubation on the conditions optimal lysis were condition tested following investigated the 3 to cycles this point, of −20 different °C/65 °C concentrations heat-shock treatment of DTT andand the its incubationincubation conditions of a mixture were of tested100–200 following mM DTT, the 200 3 cycles U/µL of Ready-Lyse−20 ◦C/65 ◦lysozyme,C heat-shock and treatment 0.5 mM EDTAand the at incubation37 °C for 2 ofh (Figure a mixture 8). ofThe 100–200 procedure mM wa DTT,s followed 200 U/µ byL Ready-Lyse the addition lysozyme, of the D2 andlysis 0.5 buffer mM andEDTA incubation at 37 ◦C forat 65 2 h °C (Figure for 28 h.). TheHowever, procedure this wasattempt followed did not by thelead addition to any detectable of the D2 lysis amplification. buffer and Inincubation spite of atthe 65 thick◦C for and 2 h. complex However, cyanobacterial this attempt did cell not wall lead structure, to any detectable we assume amplification. that the elevated In spite incubationof the thick temperature and complex of cyanobacterial the D2 buffer cellposes wall a very structure, narrow we window assume thatof optimal the elevated incubation incubation time whichtemperature is tedious of theand D2 time-consumi buffer posesng a to very pinpoint, narrow and window lowering of optimalits incubation incubation temperature time which would is broadentedious andthe window time-consuming of optimal to pinpoint,incubation and length lowering and allow its incubation for the timely temperature stopping would of the broaden reactions the priorwindow to the of optimalover-denaturation incubation of length the released and allow DNA for thetemplates. timely stoppingTherefore, of th thee D2 reactions buffer priorincubation to the temperatureover-denaturation was decreased of the released to 37 DNA °C while templates. all the Therefore, other parameters the D2 buffer remained incubation unchanged. temperature As expected,was decreased the amplified to 37 ◦C while single all cell the otherrate ramped parameters to 80% remained with unchanged.an average Asof 8.21 expected, ng of the DNA. amplified This suggestssingle cell that rate penetrating ramped to the 80% external with an layers average of Nostoc of 8.21 sp ng. ofis critical DNA. Thisfor lysozyme suggests thatto reach penetrating and digest the theexternal peptidoglycan layers of Nostoc layers.sp. is critical for lysozyme to reach and digest the peptidoglycan layers.

FigureFigure 8. 8. TheThe DTT DTT effect effect on on NostocNostoc sp.sp. lysis lysis for for SC-WGA. SC-WGA. After After the the heat heat-shock-shock treatment, treatment, a mixture a mixture of 0.5of 0.5mM mM of ofEDTA, EDTA, 200 200 U/µL U/µ ofL ofReady-Lyse Ready-Lyse lysozyme lysozyme,, and and the the DTT DTT of of different different concentrations concentrations was was ◦ addedadded to to the the cells cells and and incubated incubated at at 37 37 °CC for for 2 2 h, h, followed followed by by adding adding a a D2 D2 alkaline alkaline lysis lysis buffer. buffer. N = 10 10 singlesingle cells cells per per condition; condition; error error ba barsrs represent represent stan standarddard deviation.

AsAs an attempt toto furtherfurther increase increase the the DNA DNA amount, amount, the the DTT DTT concentration concentration was was doubled doubled to 200 to mM;200 mM;however, however, that caused that caused only a only 10% amplificationa 10% amplification rate with rate a minutewith a amountminute ofamount DNA. of Since DNA. the Since increased the increasedDTT concentration DTT concentration was expected was to expected degrade to the degrade external the layer external more layer sufficiently more sufficiently as with C. glutamicum as with C. , glutamicumthe decline, inthe the decline amplification in the amplification rate could be rate explained could be by explained the possibility by the that possibility the over-denaturation that the over- denaturation still occurred due to the extended D2 buffer incubation despite the lowered

Micromachines 2018, 9, 367 12 of 17 still occurred due to the extended D2 buffer incubation despite the lowered temperature. To test this assumption, we incubated the D2 buffer for 1 h, and the tests were repeated on the use of 100 mM and 200 mM DTT with all the other parameters unaltered. In the test with 100 mM DTT, the reduced incubation time of D2 buffer doubled the amplified DNA amount to 14.32 ng; while in the test with 200 mM DTT, 100% of amplified Nostoc sp. single-cell lysis and amplification rate and 32.7 ng of DNA was achieved, and 80% of single-cell replicates reached >25 ng DNA with statistically significant improvements compared to the prior attempt (p-value = 0.002). Despite of the fact that C. glutamicum and Nostoc sp. differ greatly in cell wall structures, it is worthwhile to mention that the chemical components of the customized lysis buffer mixture, their concentrations, and incubation temperature necessary to achieve optimal lysis condition for the subsequent DNA amplification turned out to be the same (200 U/µL Ready-Lyse lysozyme, 200 mM DTT, 0.5 mM EDTA, 37 ◦C). This shows that the customized mixture has the potential to be used without modification for lysing many other bacterial species, while its incubation time is determined by the thickness and the nature of the cell walls. Thermal treatment poses a distinctive effect on species that have thick and rigid cell walls, but may not be necessary for those with comparatively thinner cell walls. As a last step of lysis and denaturing template DNA, we recommend using the Qiagen D2 buffer at 37 ◦C or lower to pinpoint an optimal incubation time for the customized lysis buffer.

3.7. The Evaluation of the Optimized Lysis Protocol Using Gloeocapsa sp. and Sphaerocystis sp. To validate the effectiveness of this bacterial lysis method for especially rigid species in single-cell genomic studies, we tested the optimized buffer mixture and lysis conditions on another two hard-to-lyse species (Figure9). Gloeocapsa sp. was chosen as the first target due to the significant challenges of cell wall disruption, even in bulk studies. Sphaerocystis sp. was chosen as a second target because even though it is a eukaryotic algal species, its very thick extracellular matrix, mainly composed of polysaccharides, encapsulated the cells, making it equally hard to penetrate for single-cell DNA amplification in our earlier attempts. Due to these reasons, we assume that these two species would be lysed sufficiently for amplification by directly using the protocol optimized for Nostoc sp. without further efforts or modification. Each set of validation experiment was repeated three times for Nostoc sp., Sphaerocystis sp. and Gloeocapsa sp. As expected, the results show that all three species reached a 100% single-cell amplification rate and an average of 66.5 ng, 73.0 ng and 42.8 ng of DNA respectively, and 100% of single-cell replicates amplified to >25 ng DNA. However, Nostoc sp., Gloeocapsa sp. and Sphaerocystis sp. had an average of 7.8 ng, 18.1 ng, and 8.7 ng of DNA in the extracellular milieu that the cells were suspended in, but no DNA was detected in the sterile PBS after amplification. However, the amplified genomic DNA showed a reasonable quality and was easily distinguished from the extracellular milieu based on the Tapestation results (Figure 10). We concluded that there was extracellular DNA in the cell suspension which was most likely caused by the process of dissociating the cell clusters into single-cell suspensions for species that are tightly clustered. For Nostoc sp., pestling was necessary to obtain sufficient single cells; and for Gloeocapsa sp. and Sphaerocystis sp. the dissociation relied on extended pestling and mild sonication, respectively, which could lead to cell disruption. Besides, unlike pure bacterial cultures, the environmental samples would be more likely to contain contaminants. Micromachines 2018, 9, x FOR PEER REVIEW 13 of 17

Micromachines 2018, 9, 367 13 of 17 Micromachines 2018, 9, x FOR PEER REVIEW 13 of 17

Figure 9. (a) Microscopic images of Nostoc sp., Sphaerocystis sp. and Gloeocapsa sp. The black boxes Figure 9. (a) Microscopic images of Nostoc sp., Sphaerocystis sp. and Gloeocapsa sp. The black boxes Figureindicate 9. additional(a) Microscopic cells from images another of Nostoc capturesp., toSphaerocystis representativelysp. and illustrateGloeocapsa cellssp. in Thesuspension. black boxes (b) indicate additional cells from another capture to representatively illustrate cells in suspension. (b) indicateThe use of additional the developed cells fromlysis anotherprotocol captureon Sphaerocystis to representatively sp. and Gloeocapsa illustrate sp. cells for lysis in suspension. and SC-WGA. (b) The use of the developed lysis protocol on Sphaerocystis sp. and Gloeocapsa sp. for lysis and SC-WGA. The use of the developed lysis protocol on Sphaerocystis sp. and Gloeocapsa sp. for lysis and SC-WGA. N = 10N =single 10 single cells cells per per species species per per condition; condition; errorerror barsbars representrepresent standard standard deviation. deviation. Each Each set of set of N = 10 single cells per species per condition; error bars represent standard deviation. Each set of experimentexperiment was was repeated repeated three three times. times. The The sterilesterile PBS asas a a true true negative negative control control did didnot notshow show any any experimentcontaminationcontamination was after repeated after amplification amplification three times. (not (not shown Theshown sterile inin the PBS graph).graph). as a true negative control did not show any contamination after amplification (not shown in the graph).

Figure 10. The Tapestation results of amplified DNA from single Nostoc sp., Gloeocapsa sp. and Sphaerocystis sp. cells using the optimized protocol. The DNA was not degraded. Three single cells Figure 10.10. The Tapestation results of amplifiedamplified DNADNA fromfrom singlesingle NostocNostoc sp.,sp., GloeocapsaGloeocapsa sp.sp. and from each species were shown. Sphaerocystis sp. cells using the optimizedoptimized protocol.protocol. The DNA was notnot degraded.degraded. Three single cellscells from4. Conclusions eacheach speciesspecies werewere shown.shown.

4. Conclusions Single cell whole genome sequencing has found various applications in mammalian cells due to the ease of cell lysis for genome amplification. However, it has rarely been applied to microbial cells, Singleand one cell of whole the major genome hurdles sequencing is the hassufficient found lysis various of the applications multilayered in mammalian cell walls without cells due to the easecompromising of cell lysis the for integrity genome of amplification. amplification. the minute amount However, However, of DNA it has template rarely for been subsequent applied amplification.to microbial microbial cells, and oneThisone of challengeof the the major majoris hurdlesespecially hurdles is prominent the sufficientis the insufficient microfluidic lysis of thelysis multilayeredplatforms of the as multilayeredthe cell microfluidic-based walls without cell walls compromising genome without thecompromising integrityamplification of the the is integrity minuteincompatible amount of the with minute of standard DNA amount template bacterial of forcellDNA subsequentlysis template methods amplification.for and subsequent commonly Thisusedamplification. challengelytic isThis especially chemicals.challenge prominent isThis especially work in microfluidicfocused prominent on developing platformsin microfluidic ef asfective the platforms microfluidic-based bacterial as single-cell the microfluidic-based genome lysis methods amplification andgenome is performed subsequent SC-WGA in a microfluidic platform to obtain >25 ng genomic DNA sufficient incompatibleamplification withis incompatible standard bacterial with standard cell lysis methodsbacterial andcell commonlylysis methods used and lytic commonly chemicals. Thisused worklytic focusedfor ondownstream developing processing. effective bacterialBy combining single-cell therma lysisl treatment methods and and chemicals performed including subsequent lysozyme, SC-WGA chemicals.EDTA, ThisDTT andwork alkaline-based focused on buffer, developing 100% of theeffective bacterial bacterial single-cell single-cell lysis rate waslysis achieved methods for and in a microfluidic platform to obtain >25 ng genomic DNA sufficient for downstream processing. By performedboth gram-positive subsequent and SC-WGA gram-negative in a microfluidic species including platform C. glutamicum to obtain ,>25 Nostoc ng spgenomic. and Gloeocapsa DNA sufficient sp., combiningfor downstreamas well thermal as Sphaerocystisprocessing. treatment andspBy.—a chemicalscombining hard-to-lyse including therma eukaryoticl lysozyme,treatment species, EDTA,and without chemicals DTT andresorting alkaline-basedincluding to off-chip lysozyme, buffer, 100% of the bacterial single-cell lysis rate was achieved for both gram-positive and gram-negative EDTA, DTT and alkaline-based buffer, 100% of the bacterial single-cell lysis rate was achieved for speciesboth gram-positive including C. and glutamicum gram-negative, Nostoc speciessp. including and Gloeocapsa C. glutamicumsp., as, wellNostoc as spSphaerocystis. and Gloeocapsasp.—a sp., hard-to-lyseas well as eukaryoticSphaerocystis species, sp.—a without hard-to-lyse resorting eukaryotic to off-chip conventionalspecies, without steps, resorting polymerase-inhibitive to off-chip reagents or multiple rounds of amplification. Even though species may react differently to the same

Micromachines 2018, 9, 367 14 of 17 lysis protocol, a combination of three primary lysis treatments applicable in microfluidic platforms that degrade bacterial cell walls in their distinct ways offers a baseline for adapting the parameters to reach optimal conditions for species of interest. This work addressed one of the major obstacles of applying SC-WGS technologies to the microbial cells by providing a single-cell lysis guideline that can be adapted to facilitate the lysis and amplification of various types of bacterial and algal cells that are challenging for single-cell genome analyses in microfluidic systems. Ultimately, we envision that it would be possible to perform single-cell genomic studies on a vast range of microbial cells in various research including environmental studies and the genomic investigation of rapidly-growing and deadly but multi-drug resistant microbial pathogens such as ESKAPE [78](Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) using microfluidic platforms and would potentially lead to the discovery of effective therapies.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-666X/9/8/367/s1. Author Contributions: Conceptualization, Y.L. and M.W.-A.; Formal analysis, Y.L.; Funding acquisition, M.W.-A.; Investigation, Y.L. and M.W.-A.; Methodology, Y.L. and M.W.-A.; Project administration, D.S.-M., J.-P.d.V., C.C., T.L., M.B. and M.W.-A.; Resources, D.S.-M., J.-P.d.V., C.C., T.L., M.B. and M.W.-A.; Supervision, D.S.-M., J.-P.d.V., C.C., T.L., M.B. and M.W.-A.; Validation, Y.L.; Writing–original draft, Y.L.; Writing–review & editing, D.S.-M., J.-P.d.V., C.C., T.L., M.B. and M.W.-A. Funding: This research was funded by the following sources. Marina Walther-Antonio and Yuguang Liu acknowledge The Ivan Bowen Family Foundation and CTSA Grant Number KL2 TR002379 from the National Center for Advancing Translational Science (NCATS). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Dirk Schulze-Makuch and Jean-Pierre de Vera acknowledge support from ERC Advanced Grant “HOME” (# 339231), and in addition, Jean-Pierre de Vera acknowledges support from ESA for the BIOMEX project (ESA-ILSRA 2009-0834) and DLR for the DLR-FuW-Project BIOMEX (2474128). Charles S. Cockell was supported by the Science and Technology Facilities Council (STFC), Grant No. ST/M001261/1. Acknowledgments: We thank the Mayo Clinic Microbiome Program and the Mayo Clinic Center for Individualized Medicine for support. We also thank Minnesota Nano Center at the University of Minnesota (Minneapolis, MN, USA) and Alexander Revzin at Mayo Clinic for granting us access to their microfabrication facilities. Conflicts of Interest: The authors declare no conflict of interest.

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