MicrobiomeAnalysis
MethodsandProtocols
Editedby RobertG.Beiko
FacultyofComputerScience,DalhousieUniversity,Halifax,NS,Canada
WillHsiao
DepartmentofPathology&LaboratoryMedicine,UniversityofBritishColumbia, Vancouver,BC,Canada
JohnParkinson
PrograminMolecularMedicine,TheHospitalforSickChildren,Toronto,ON,Canada; DepartmentofBiochemistryandDepartmentofMolecularGenetics,UniversityofToronto, Toronto,ON,Canada
Editors
RobertG.Beiko
FacultyofComputerScience DalhousieUniversity Halifax,NS,Canada
JohnParkinson
PrograminMolecularMedicine
TheHospitalforSickChildren Toronto,ON,Canada
DepartmentofBiochemistry andDepartmentofMolecularGenetics UniversityofToronto Toronto,ON,Canada
WillHsiao
DepartmentofPathology &LaboratoryMedicine
UniversityofBritishColumbia Vancouver,BC,Canada
ISSN1064-3745ISSN1940-6029(electronic)
MethodsinMolecularBiology
ISBN978-1-4939-8726-9ISBN978-1-4939-8728-3(eBook) https://doi.org/10.1007/978-1-4939-8728-3
LibraryofCongressControlNumber:2018955280
© SpringerScience+BusinessMedia,LLC,partofSpringerNature2018
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Preface
Themicrobiome,coinedbyLederbergandMcCrayas“...theecologicalcommunityof commensal,symbiotic,andpathogenicmicroorganismsthatliterallyshareourbodyspace” [1],drawstogetheraremarkablenumberofdisciplineswiththeoverarchinggoalof understandingandultimatelyharnessingtheworkingsofmicrobialsystems.Truetothe initialconceptionoftheterm,thehumanmicrobiomecontinuestobeintensivelystudied, butmicrobialsampleshavebeencollectedfromnearlyeveryimaginablehabitatonEarth, fromtheupperatmospheretotheseabedsubsurface,fromhotspringstoglacierice,and fromnematodegutstowhalecarcasses.
Microbiomeanalysismakesfrequentuseofacommonsetoftoolstoaddressmany pertinentquestions.Acommonworkflowformicrobiomeanalysislookssomethinglikethis: collectsample(e.g.,soil,water,stool),extractDNA,performDNAsequencing,anduse bioinformaticstoolstodescribeimportantpropertiesofthemicrobiome.Thispipelinehas beenappliedtohugenumbersofsamplesfromadiversearrayofenvironments.Inparticular, thetargetedsequencingviapolymerasechainreaction(PCR)amplificationof“marker” genesthatareseenasdiagnosticofdifferenttypesofmicroorganismshasseenwidespread use.Theworkhorseofmicrobialdiversityhasthusfarbeenthe16SribosomalRNAgene, whichhasbeenthesubjectofintensiveprotocoldevelopment:seeforexampletheEarth MicrobiomeProjectprotocols[2],andadetailedevaluationof16Ssequencingonthe Illuminasequencingplatforms[3].However,whilecapturingthe taxonomiccomposition ofa microbialcommunity,marker-genesequencingislimitedinitsabilitytorevealthediversity of functions present,requiringtheapplicationofalternativeapproaches.
Gaininganaccurateandrelevantpictureofthemicrobiomerequirescarefulexperimentaldesign,andthefirstpartofourbookfocusesontheprofilingofdifferenthabitatsand elementsofbiodiversity.Theprocedurestocollectrepresentativeanduncontaminated samplescanbehighlycomplex;oneneedlooknofurtherthanChapter 1 foranexample ofthechallengesassociatedwithcollectingmicrobialsamplesfromthedeepsubsurface. DNAsequencingmightbeseenasthefoundationofmicrobialcommunityanalysis,evenif arguablythefirstsuchanalysiswasdonewithRNAratherthanDNAsequencinginthe famousOctopusSpringstudy[4].However,other“meta-omic”methodsthatconsider messengerRNAtranscripts,proteinproducts,ormetabolitelevelscanrevealagreatdeal moreaboutmicrobialactivitiesinaparticularhabitat.Thecombinationofmultiplesuch strategiescanbeespeciallypowerful,asillustratedbytheuseofDNAsequencingtosupport targetedmetaproteomics(Chapter 6).
Theremainderofthisvolumeaddressesthecomputationalchallengesofmicrobiome analysis.Animmensenumberofalgorithmsandsoftwarepackageshavebeendevelopedfor thetask,andevenseeminglysimplequestionsas“whatisthebiodiversitypresentinagiven sample?”maynotbestraightforward,asexemplifiedbyChapter 10.Atthesametime,the richinformationgeneratedfromthesesamplesisdrivingthedevelopmentofinnovative toolsandpipelineswiththeabilitytogeneratenoveldatatypesandaddressnewquestions, suchastherecoveryofcompletegenomesfrommetagenomes(Chapter 14),andtheuseof networkapproachestoidentifypatternsofmicrobialassociation(Chapter 17).
Althoughnobookonmicrobiomeanalysiscanbeexhaustive,inpreparingourvolume wehavesoughttoconveywhatmightbeseenasstandardpracticeinthefield(totheextent
anythingcanbeclaimedassuch!),whilealsohighlightingtechniquesatthefrontiersofthe fieldthatchallengestandardpractice.Reflectingthecontinueddominanceofmarker-gene approaches,theQIIMEpackage[5]recentlyreceiveditsten-thousandthcitation:therecent releaseofthesecondversionofthissoftwareisnotablebecauseitisdevelopedina completelydifferentframework,andbecauseitupendssomeofthetoolsandtechniques thathaveheretoforeservedasitsdefaults(see Chapter 8).
Bydefiningproceduresinpreciseterms,the MethodsinMolecularBiology seriescontributestothereproducibilityofexperiments.However,reproducibilityinbioinformaticsis abigconcern[6],withseveralmovingpartsincludingdatabaseversions,softwareupdates, andparametersettings.Comparingnewmethodstoexistingonesdemandsthatfinalresults andallintermediatestepscanberegenerated.Thelastfewyearshaveseensignificant advancesinreproducibilitythroughmeanssuchasautomatedworkflowtoolsincluding Galaxy,interactivecodetoolssuchasJupyterNotebooks,andrepositorieswithversion control,themostnotableexampleofwhichiscurrentlyGithub.Wearepleasedthatmanyof ourauthorshaveprovidedexamplesthatmakeuseofthesetools,whichwillmakeit considerablyeasierforreaderstoperformanalysesinaconsistentmanner.
Itremainsonlyforustothanktheindividualswhohavecontributedtheirtimeandhard worktopreparingahighlydiverseandengagingsetofchapters.WearealsogratefultoJohn Walkerfortheoriginalinvitationtopreparethisbook.
Halifax,NS,CanadaRobertG.Beiko Vancouver,BC,CanadaWillHsiao Toronto,ON,CanadaJohnParkinson
References
1. LederbergJ,McCrayAT(2001)OmeSweetOmics—agenealogicaltreasuryofwords. Scientist15;8
2. EarthMicrobiomeProject.ProtocolsandStandards. http://www.earthmicrobiome. org/protocols-and-standards/.Accessed3March2018
3. CaporasoJG,LauberCL,WaltersWA,etal(2012)Ultra-high-throughputmicrobial communityanalysisontheIlluminaHiSeqandMiSeqplatforms.ISMEJ6:1621
4. StahlDA,LaneDJ,OlsenGJ,etal(1985)CharacterizationofaYellowstonehotspring microbialcommunityby5SrRNAsequences.ApplEnvironMicrobiol49:1379–1384
5. CaporasoJG,KuczynskiJ,StombaughJetal(2010).QIIMEallowsanalysisofhighthroughputcommunitysequencingdata.NatMethods7:335
6. SandveGK,NekrutenkoA,TaylorJetal(2013)Tensimplerulesforreproducible computationalresearch.PLoSComputBiol9:e1003285
Preface.. ...................................................................v Contributors
1CharacterizingtheDeepTerrestrialSubsurfaceMicrobiome.. ...............1 RebeccaA.Daly,KellyC.Wrighton,andMichaelJ.Wilkins
2FreshwaterViromes:FromSamplingtoEvaluation.. .......................17 CatherinePutonti,Zoe Diener,andSiobhanC.Watkins
3CharacterizationofEukaryoticMicrobiomeUsing18S AmpliconSequencing ...................................................29 AnaPopovicandJohnParkinson
4CultureandMolecularProfilingoftheRespiratoryTractMicrobiota.. ........49 FionaJ.Whelan,LauraRossi,JenniferC.Stearns, andMichaelG.Surette
5MethodsandStrategiestoExaminetheHumanBreastmilkMicrobiome ......63 LaurenLeMay-Nedjelski,JuliaCopeland,PaulineW.Wang, JamesButcher,SharonUnger,AlainStintzi,andDeborahL.O’Connor
6QuantificationofVitaminB12-RelatedProteinsinMarine MicrobialSystemsUsingSelectedReactionMonitoring MassSpectrometry.. ....................................................87 ErinM.Bertrand
7Single-CellGenomicsofMicrobialDarkMatter ............................99 ChristianRinke
816SrRNAGeneAnalysiswithQIIME2 ...................................113 MichaelHallandRobertG.Beiko
9Processinga16SrRNASequencingDatasetwiththeMicrobiome HelperWorkflow........................................................131 GavinM.Douglas,Andre´M.Comeau,andMorganG.I.Langille
10NormalizationofMicrobiomeProfilingData. ..............................143 PaulJ.McMurdie
11PredictingtheFunctionalPotentialoftheMicrobiome fromMarkerGenesUsingPICRUSt. .....................................169 GavinM.Douglas,RobertG.Beiko,andMorganG.I.Langille
12MetagenomeAssemblyandContigAssignment ............................179 QingpengZhang
13FromRNA-seqtoBiologicalInference:UsingCompositional DataAnalysisinMeta-Transcriptomics ....................................193 JeanM.MacklaimandGregoryB.Gloor
14SubsampledAssembliesandHybridNucleotide Composition/DifferentialCoverageBinningforGenome-Resolved Metagenomics ..........................................................215 LauraA.Hug
15TranskingdomNetworks:ASystemsBiologyApproachtoIdentify CausalMembersofHost–MicrobiotaInteractions... .......................227
RichardR.Rodrigues,NataliaShulzhenko,andAndreyMorgun
16ConstructingandAnalyzingMicrobiomeNetworksinR ....................243 MehdiLayeghifard,DavidM.Hwang,andDavidS.Guttman
17BayesianInferenceofMicrobialCommunityStructure fromMetagenomicDataUsingBioMiCo ..................................267
KatherineA.Dunn,KatelynAndrews,RanaO.Bashwih, andJosephP.Bielawski
18AnalyzingMetabolicPathwaysinMicrobiomes .............................291 MobolajiAdeolu,JohnParkinson,andXuejianXiong
19SparseTreatment-EffectModelforTaxonIdentification withHigh-DimensionalMetagenomicData.. ..............................309 ZhenqiuLiuandShiliLin
Index... ...................................................................319
Contributors
MOBOLAJI ADEOLU PrograminMolecularMedicine,TheHospitalforSickChildren, Toronto,ON,Canada
KATELYN ANDREWS DepartmentofMathematicsandStatistics,DalhousieUniversity, Halifax,NS,Canada
RANA O.BASHWIH DepartmentofMathematicsandStatistics,DalhousieUniversity, Halifax,NS,Canada
ROBERT G.BEIKO FacultyofComputerScience,DalhousieUniversity,Halifax,NS, Canada
ERIN M.BERTRAND DepartmentofBiology,DalhousieUniversity,Halifax,NS,Canada
JOSEPH P.BIELAWSKI DepartmentofMathematicsandStatistics,DalhousieUniversity, Halifax,NS,Canada
JAMES BUTCHER OttawaInstituteofSystemsBiology,Ottawa,ON,Canada;Departmentof MicrobiologyandImmunology,UniversityofOttawa,Ottawa,ON,Canada
ANDRE ´ M.COMEAU IntegratedMicrobiomeResource,DalhousieUniversity,Halifax,NS, Canada
JULIA COPELAND CentrefortheAnalysisofGenomeEvolutionandFunction,Universityof Toronto,Toronto,ON,Canada
REBECCA A.DALY DepartmentofMicrobiology,TheOhioStateUniversity,Columbus,OH, USA
ZOE DIENER DepartmentofBiology,NewMexicoInstituteforMiningandTechnology, Socorro,NM,USA
GAVIN M.DOUGLAS DepartmentofMicrobiologyandImmunology,DalhousieUniversity, Halifax,NS,Canada
KATHERINE A.DUNN DepartmentofMathematicsandStatistics,DalhousieUniversity, Halifax,NS,Canada
GREGORY B.GLOOR DepartmentofBiochemistry,SchulichSchoolofMedicineand Dentistry,TheUniversityofWesternOntario,London,ON,Canada;CanadianCentre forHumanMicrobiomeandProbioticResearch,LawsonHealthSciencesCentre,London, ON,Canada
DAVID S.GUTTMAN DepartmentofCellandSystemsBiology,UniversityofToronto,Toronto, ON,Canada;CentrefortheAnalysisofGenomeEvolutionandFunction,Universityof Toronto,Toronto,ON,Canada
MICHAEL HALL DalhousieUniversity,Halifax,NS,Canada
LAURA A.HUG DepartmentofBiology,UniversityofWaterloo,Waterloo,ON,Canada
DAVID M.HWANG DepartmentofPathology,UniversityHealthNetwork,Toronto,ON, Canada;DepartmentofLaboratoryMedicineandPathobiology,UniversityofToronto, Toronto,ON,Canada
MORGAN G.I.LANGILLE DepartmentofMicrobiologyandImmunology,Dalhousie University,Halifax,NS,Canada;IntegratedMicrobiomeResource,DalhousieUniversity, Halifax,NS,Canada;DepartmentofPharmacology,DalhousieUniversity,Halifax,NS, Canada
MEHDI LAYEGHIFARD DepartmentofCellandSystemsBiology,UniversityofToronto, Toronto,ON,Canada
LAUREN LEMAY-NEDJELSKI FacultyofMedicine,DepartmentofNutritionalSciences, UniversityofToronto,Toronto,ON,Canada;TranslationalMedicine,TheHospitalfor SickChildren,Toronto,ON,Canada
SHILI LIN DepartmentofStatistics,TheOhioStateUniversity,Columbus,OH,USA
ZHENQIU LIU SamuelOschinComprehensiveCancerInstitute,Cedars-SinaiMedical Center,LosAngeles,CA,USA
JEAN M.MACKLAIM DepartmentofBiochemistry,SchulichSchoolofMedicineandDentistry, TheUniversityofWesternOntario,London,ON,Canada;CanadianCentreforHuman MicrobiomeandProbioticResearch,LawsonHealthSciencesCentre,London,ON,Canada
PAUL J.MCMURDIE WholeBiome,Inc.,SanFrancisco,CA,USA
ANDREY MORGUN CollegeofPharmacy,OregonStateUniversity,Corvallis,OR,USA
DEBORAH L.O’CONNOR FacultyofMedicine,DepartmentofNutritionalSciences, UniversityofToronto,Toronto,ON,Canada;TranslationalMedicine,TheHospitalfor SickChildren,Toronto,ON,Canada;DepartmentofPediatrics,MountSinaiHospital, Toronto,ON,Canada
JOHN PARKINSON PrograminMolecularMedicine,TheHospitalforSickChildren,Toronto, ON,Canada;DepartmentofBiochemistryandDepartmentofMolecularGenetics, UniversityofToronto,Toronto,ON,Canada
ANA POPOVIC PrograminMolecularMedicine,TheHospitalforSickChildren,Toronto, ON,Canada;DepartmentofBiochemistry,UniversityofToronto,Toronto,ON,Canada
CATHERINE PUTONTI DepartmentofBiology,LoyolaUniversityChicago,Chicago,IL,USA; DepartmentofComputerScience,LoyolaUniversityChicago,Chicago,IL,USA; BioinformaticsProgram,LoyolaUniversityChicago,Chicago,IL,USA;Departmentof MicrobiologyandImmunology,StritchSchoolofMedicine,LoyolaUniversityChicago, Maywood,IL,USA
CHRISTIAN RINKE AustralianCentreforEcogenomics,UniversityofQueensland,Brisbane, QLD,Australia
RICHARD R.RODRIGUES CollegeofPharmacy,OregonStateUniversity,Corvallis,OR,USA
LAURA ROSSI DepartmentofBiochemistryandBiomedicalSciences,McMasterUniversity, Hamilton,ON,Canada
NATALIA SHULZHENKO CollegeofVeterinaryMedicine,OregonStateUniversity,Corvallis, OR,USA
JENNIFER C.STEARNS DepartmentofMedicine,McMasterUniversity,Hamilton,ON, Canada
ALAIN STINTZI OttawaInstituteofSystemsBiology,Ottawa,ON,Canada;Departmentof MicrobiologyandImmunology,UniversityofOttawa,Ottawa,ON,Canada
MICHAEL G.SURETTE DepartmentofMedicine,McMasterUniversity,Hamilton,ON, Canada
SHARON UNGER TranslationalMedicine,TheHospitalforSickChildren,Toronto,ON, Canada;FacultyofMedicine,DepartmentofPediatrics,UniversityofToronto,Toronto, ON,Canada;DepartmentofPediatrics,MountSinaiHospital,Toronto,ON,Canada
PAULINE W.WANG CentrefortheAnalysisofGenomeEvolutionandFunction,Universityof Toronto,Toronto,ON,Canada
SIOBHAN C.WATKINS DepartmentofBiology,NewMexicoInstituteforMiningand Technology,Socorro,NM,USA
FIONA J.WHELAN DepartmentofBiochemistryandBiomedicalSciences,McMaster University,Hamilton,ON,Canada
MICHAEL J.WILKINS DepartmentofMicrobiology,TheOhioStateUniversity,Columbus, OH,USA
KELLY C.WRIGHTON DepartmentofMicrobiology,TheOhioStateUniversity,Columbus, OH,USA
XUEJIAN XIONG PrograminMolecularMedicine,TheHospitalforSickChildren,Toronto, ON,Canada
QINGPENG ZHANG DepartmentofEnergy,JointGenomeInstitute,WalnutCreek,CA,USA
CharacterizingtheDeepTerrestrialSubsurfaceMicrobiome
RebeccaA.Daly,KellyC.Wrighton,andMichaelJ.Wilkins
Abstract
Alargeportionoftheearth’sbiomassresidesinthesubsurfaceandrecentstudieshaveexpandedour knowledgeofindigenousmicrobiallife.Advancesinthefieldofmetagenomicsnowallowanalysisof microbialcommunitiesfromlow-biomasssamplessuchasdeep(>2.5km)shalecoresamples.Herewe presentprotocolsforthebestpracticesincontaminationcontrol,handlingcorematerial,extractionof nucleicacids,andlow-inputlibrarypreparationforsubsequentmetagenomicsequencing.
Keywords Deeplife,Deepbiosphere,Terrestrialsubsurface,Contamination,Shale,Metagenomics, Lowbiomass
1Introduction
Whileitisestimatedthattheterrestrialsubsurfaceisthelargest reservoiroflifeonEarth,hostingbetween40%and60%ofall bacterialcells,densitiesaretypicallylowindeepsubsurfaceecosystems[1, 2].Researchintothedeepterrestrialbiospherestemsfrom interestinbiogeochemicalcyclingandthediscoveryofnovelbiodiversityandmetabolisms[3].However,duetothedifficultyin obtainingcoresamplesfromthousandsofmetersbelowtheEarth’s surfaceandcharacteristiclowbiomass,itiscriticalthatsamplesare collectedandpreservedinamannertolimitcontamination. Advancesinmetagenomicsequencingtechnologywhichpermits libraryconstructionfrompicogramquantitiesofDNAcannowbe utilizedtoexaminenotonlythepresenceofsingle-genemarkers (i.e.,16SrRNAgenes),butallowforreconstructionofentire microbialgenomes,providinginsightintofunctionalpotential withinthedeepterrestrialsubsurfacemicrobiome.
Thereisalargebodyofpublishedworkdetailinghowtodeal withcontaminationoflow-biomasssamplesduringdrillingand coring,andthesourcesofcontamination[3–6].Smallamountsof contaminatingbacteriacanmaskthesignalfromindigenous
RobertG.Beikoetal.(eds.), MicrobiomeAnalysis:MethodsandProtocols,MethodsinMolecularBiology,vol.1849, https://doi.org/10.1007/978-1-4939-8728-3_1, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2018
2Materials
2.1Collection andDNAExtraction ofDrillingMuds andOtherFluids
microorganismsandcompromisefutureanalyses.Methodsoften includetheuseoftracersduringthedrillingprocess,including microbialtracers(e.g.,livecells)[7],chemicaltracerssuchasperfluorocarbons[8],andvisualtracerssuchasfluorescein[9]and fluorescentmicrospheres[4, 10–13].Thecollectionof“blanks”at multiplepointsofsampleprocessingprovidestheabilitytodistinguishcontaminatingmicroorganismsfromindigenouslife.Once contaminationcontrolshavebeenimplemented,thequestionstill remainsofhowtoextractDNAfromlow-biomassrockmatrices.
DNAiscommonlyextractedbyusingacombinationofchemicalandphysicallysis[14–18]inordertorecoverandsubsequently purifyDNAfromlysedcells,usablefordownstreammolecular assays.ThereareawiderangeofDNA-extractionprotocols,with certainmethodsoptimizedforparticularsampletypes,including commercialkits.Forchallenginganduniquesamples,theremay benoestablishedmethods.Thereisno“one-size-fits-all”DNAextractionprotocol,anditisrecommendedthatmultiplemethods aretestedandcomparedonsamplematerialphysicallyandchemicallysimilartothetargetedsamples.OnceDNAhasbeensuccessfullyextractedandpurifiedfromthematrix,commercialkitsare availableforlow-inputlibrarypreparationformetagenomic sequencing[19, 20].
Thischapteroutlinesproceduresforcontaminationcontrolin thefield,theuseoffluorescentmicrospheretracersduringdrilling, contaminationcontrolinthelab,sampledecontamination/cleaning,samplegrinding,DNAextractionandlibrarypreparationfor metagenomicsequencingforcoresamplesobtainedfromtheMarcellusshaleformationinWestVirginia,USA.
1.Nalgene1Lwide-mouthHDPEbottles,autoclaved.
2.0.2 μmpolyethersulfone(PES)vacuumfilterdevice.
3.MoBioPowerMaxSoilDNAisolationkit(MoBioLaboratories,Carlsbad,CA,USA).
4.StandardPCRreagentsandprimersfor16SrRNAgene amplification.
2.2Fluorescent Microspheres
1.FluoresbriteYGcarboxylatemicrospheres0.50 μm(Polysciences,Warrington,PA,USA).
2.Sterilecarboyorothersimilarcontainerfilledwithdeionized waterwithasensitivityof18MΩ cmat25 C.
2.3CoreCollection
1.Whirl-Pakbags(Nasco,FortAtkinson,WI,USA)oftheappropriatesizetoholdasinglecore.
2.Anaeropak7.0Lrectangularjars(Mitsubishi,Tokyo,Japan).
3.AnaeroPouchSystem-AnaeroAnaerobicGasGeneratorsachets (Mitsubishi,Tokyo,Japan).
2.4Quantification ofMicrospheres
2.5CoreSample Decontamination
2.6GrindingofCore Samples
1.Inverted,fluorescentmicroscopewitha10 objective(for 100 totalmagnification).Ensurethatthemicroscopehas enoughclearancetoallowthecoresampletobeplacedon thestage.WeusedanEclipseTiinvertedmicroscope(Nikon, Tokyo,Japan).
2.Softwareforobtainingcolormicrographs.
1.1.5MNaClsolution:87.66gofNaCl.AddDNA-freewaterto avolumeof1L.Mixandautoclave.
2.Autoclavedaluminumdishwithflutedsides, 200mL(volumedependentonthesizeofcoresamples).
3.Autoclavedhighquality,fine(size00)steelwool.Autoclaved steelwoolwrappedinloosealuminumfoil.Afterautoclaving dryina50 Coventopreventrusting.
1.Bunsenburner.
2.Metaltongsforholdingcoresamples.
3.Autoclaved12 12”sheetsofaluminumfoil.
4.Cleaned,autoclavedceramicmortarsandpestles.
5.BrassmeshsieveswithSSwire,includingsievecoverand bottom(e.g.,stackable3”sievesin#10,#18,and#35mesh (HumboldtManufacturingCompany,Elgin,IL,USA)).
6.Plattner’shardenedsteelmortarsandpestles(Humboldt ManufacturingCompany,Elgin,IL,USA).
7.Coldchisel,½”cutwidth.
8.Sledge/drillinghammerwithcompacthandle( 3lb. hammer).
2.7DNAExtraction fromGroundCore
1.DNAZapPCRDNADegradationsolutions(ThermoFisher Scientific,Waltham,MA,USA).
2.LysisBufferI,pH10,250mL:Add17.5mLof1.0M,pH7.5 Tris–HCl;15.0mLof0.5M,pH8.0EDTA,25.0mLof8.0M guanidinehydrochloride,and1.25mLofTritonX-100toa sterileflask.AddDNA-freewatertoavolumeof250mL.Mix andadjustpHto10.0withNaOH.Filtersterilizethrougha 250mL,0.1 μm,PESvacuumfilterunit.
3.DNALoBind1.5mLtubes(Eppendorf,Hauppauge,NY, USA).
2.8DNA Quantification
2.9Library Preparation forMetagenomic Sequencing andOptionalMDA Amplification
4.Ultra-high-speed50mLcentrifugetubes(VWRInternational, Radnor,PA,USA).
5.Phenol:chloroform:isoamylalcohol,25:24:1,pH8.0.
6.Chloroform:isoamylalcohol,24:1.
7.100%ethanol.
8.70%ethanol,preparedwithDNA-freewater.
9.EBbuffer(Qiagen,Valencia,CA,USA).
10.Linearacrylamide,5mg/mL(ThermoFisherScientific,Waltham,MA,USA).
1.Qubitfluorometer(ThermoFisherScientific,Waltham,MA, USA).
2.QubitdsDNAHS(highsensitivity)assaykit(ThermoFisher Scientific,Waltham,MA,USA).
3.Qubitassaytubes,0.5mL(ThermoFisherScientific,Waltham, MA,USA).
1.REPLI-gSingleCellWGAkit(Qiagen,Valencia,CA,USA).
2.NexteraXTDNAlibrarypreparationkit(Illumina,Inc.,San Diego,CA,USA).
3.NexteraXTIndexkit(Illumina,Inc.,SanDiego,CA,USA).
4.AgencourtAMPureXPmagneticbeads(BeckmanCoulterLife Sciences,Indianapolis,IN,USA).
5.Magneticstandfor1.5mLmicrocentrifugetubes.
6.BioanalyzerInstrument(AgilentTechnologies,SantaClara, CA,USA).
7.BioanalyzerHighSensitivityDNAkit(AgilentTechnologies, SantaClara,CA,USA).
3Methods
3.1Contamination ControlintheField
Recoveringcorematerialfromthesubsurfacerequiresdrilling technologieswhichcanintroducecontaminationfromseveral sources,includingdrillingmudadditives,surfacewatermixed withdrillingmuds,contaminationfrommudtanks,pumping equipment,andcontaminationfromoverlyingformationsand groundwater.Inordertoobtainreliableinformationaboutindigenousmicroorganisms,itisextremelyimportanttosampleall sourcesofpotentialcontamination,extractDNA,andsequence thesesamplesforsubtractiveanalysis.Chemicalandparticletracers arecommonlyusedtoassesssampleintegrity.Herewedescribeuse offluorescentmicrospheresasavisualtracer,andDNAextraction ofthefluidsusedtocleancoresamplesasamoleculartracer,to ensuresampleintegrity.
3.1.1SamplingDrilling MudsandOtherFluids
3.1.2Addition ofFluorescent Microspheres toDrillingMuds
1.Sampleallfluidsthatcanpotentiallygodown-well,beforeand duringthedrillingprocessstarts,includingfreshwateraddedto drillingmuds,andthedrillingmuds(see Note1).
2.Ensurethatallsamplingequipmentissterile,andweardisposableglovesatalltimes.Itisusefultohavelargeladlesand bucketsonhandthatareeasilysterilizedwithableachsolution or70%ethanol,forretrievingsamplesatthewellpad.
3.Filterfreshwatersourcesthroughasterile,large-volumefilter apparatuswitha0.2 μmpolyethersulfone(PES)membrane, filteringaminimumof3LofeachsampleforfutureDNA extractionandanalysis.Ifthesamplescannotbefilteredinthe field,collectsamplesinsterile1LNalgenecontainers,orlarger carboys,filledtothebrimtominimizeheadspaceandthus alterationofthemicrobialcommunityandtransportonice.If thesamplescanbefilteredinthefield,havedryiceonhandto rapidlyfreezethefiltersduringtransporttothelaboratory.
4.Collectdrillingmudsin1LsterileNalgenecontainers.
5.DNAfromfreshwatersourcefilters( 5goffiltermaterial)and aliquotsofdrillingmuds( 5mL)canbeextractedusingthe manufacture’srecommendedprotocolusingthePowerMax SoilDNAisolationkit(see Note2).
6.TestforPCRamplificationusing“universal”16SrRNAgene primerstoensurethatsamplesaresuitablefordownstream analyses(metagenomiclibrarypreparation).
Duringthedrillingprocess,drillingmudsandotherfluidscan penetratecoresamples.WeusedFluoresbritecarboxylateyellowgreenfluorescentpolystyrenemicrospheres0.50 μmindiameteras aproxyforcontaminatingbacterialcells,andquantifiedmicrospherepenetrationandremovalduringthedecontaminationprocessbyfluorescencemicroscopy.
1.Inordertocalculatetheamountofmicrospherestoaddtothe drillingmud,itisnecessarytodeterminethevolumeofthe drillingmudinthetank(see Note3).Drillingmudsare containedinlargemixingtanks,wheretheliquidisconstantly beingmixedtomaintainhomogeneityandallowfortheadditionofdrillingmudchemicalsasrequiredbytheoperator.
2.Calculatethevolumeoffluorescentmicrospheresneededto obtainaconcentrationof 5 105 microspheres/mLinthe drillingmudtank.
3.Inordertoensurethatthemicrospheresaredispersedevenly throughoutthedrillingmud,firstdilutetherequiredvolume ofmicrospheresinacarboyorothersimilarsterilecontainer containingdeionizedwaterwithasensitivityof18MΩ cmat 25 C.
3.1.3CoreCollection
4.Mixwellandaddtothemudtankwhilethedrillingmudis beingmixed.
5.Aftermixing,sampledrillmudsformicrospherequantification andDNAextraction(see Note4).
Coresfromblackshaleformationscontainhighamountsofhydrocarbonsandextremelylowbiomass.Inordertoobtainsamples adequateformetagenomicanalyses,rigoroussteriletechnique, properstorage/transportconditions,andminimaltimelagbefore processingsamplesarecritical.
1.Beonsiteduringtheactualcollectionofcoresamples.Arrange withtheoperatoron-sitetohaveaccesstocoresimmediately.
2.LabelWhirl-Pakbagswithpertinentcoreinformation,includingthewell,depth,andtimeofsampling.
3.PhotographeachcorebeforeinsertinginaWhirl-Pakbag (dimensionsdependentoncoresize),assomecoresarerecoveredintact,whileothersarerecoveredinsmallpieces.
4.Ifthereservoirispressurized,itmaybenecessarytopierceeach Whirl-Pakbagwithathin(25G)needlesothatsamplebagsdo notburstduetode-gassingofthecores.
5.PlaceWhirl-PakbagswithcoresinAnaeropak7.0Lrectangular jars,with3,O2-consumingsachetsineachjar.
6.Transportcoresinanaerobicboxesonice,backtothelaboratoryasquicklyaspossible.
3.2Contamination ControlintheLab
3.2.1Quantification ofMicrospheresPrior toDecontamination
Minimizetimeatroom-tempandfreeze-thawcycles.Processsamplesasquicklyaspossible,storingat4 Cduringdailyprocessing stepsandstoregroundsamplesat 80 Cforlong-termstorage.
1.Carefullywipethemicroscopestagewith70%ethanol.
2.Drillmudsampleswithmicrospherescanbequantifiedby putting10–20 μLonaglassslidecoveredbyaglasscoverslip.
3.Placethecoresampleonthemicroscopestage,beingcarefulto ensurenodrillingmudorcoreparticlescontacttheobjectives. Microspheresoncoresamplescanbeenumeratedbytakingat leastfourimages,oneoneachendofthecore,andtwoimages oneachside.
4.Imagescanbesavedusingtheappropriatesoftwareforthe microscope,andcountedatalaterdate,tominimizethetime thesampleisexposedtotheair.
5.Foreachimage,countthenumberofmicrospheresineachfield ofview(ormultiple,smallerwindowsfromeachimage).
3.2.2CoreSample Decontamination
3.2.3Quantification ofMicrospheresPrior toDecontamination
6.Cleanthemicroscopestagewith70%ethanolbetweeneach sample.
7.Placingautoclavedaluminumfoilonthestagewithanopening fortheobjectivehelpskeepthemicroscopestagecleanand preventscross-contamination.
Therearemanydescribedmethodsfordecontaminationofcore samples,includingparingviacircularsawswithhydrauliccrushing, andhammerand/orchisel.Herewedeterminedthatthebest resultswereobtainedfromthreesuccessiveNaClwasheswhile scrubbingwithfinesteelwool.Duetotherelativelysoftnatureof theshalesamplesfromourstudies,thisresultedinremovalofathin layeroftheexterior,pastthepointofpenetrationofmicrospheres, andthusmicrobialcontamination.
1.Performallworkinalaminarflowhooddedicatedtocore samples.
2.Wipeallsurfaces,equipment,andpipetteswith70%ethanol.
3.Putallreagents,supplies,andpipettesinthelaminarflow hood,UVsterilizefor30min.Rotatepipettesandsupplies, UVsterilizeforanadditional30min.
4.Placethreeweighdishesinthelaminarflowhoodandaliquot 50mLof1.5MNaClsolutionineachdish.
5.Placeasmallpiece( 2” 2”)ofsteelwoolineachdish.
6.Placethecoresampleinthefirstdish,andwhilewettingthe steelwool,scrubtheexteriorofthecoresamplewithsmall circularmotions.Youmaynoticesloughedoffcoreparticlesin theNaClsolution.
7.Removethecorefromthefirstwash,andplaceitinthesecond dish.Repeatscrubbingtheexteriorofthecoresamplewiththe freshNaClsolutionandnewsteelwool.Repeattheprocessin thethirdwash.
8.Photographthecoresampleafterthedecontamination/cleaningprocessfordocumentationpurposes.
9.Placethecoreinanew,labeled,Whirl-Pakbag.
RepeattheprocessinSubheading 3.2.1,takingimagesofthe cleanedcoresurface.Itislikelythatnomicrosphereswillbe detectedafterthedecontaminationprocedure.Ifmicrospheres areobserved,repeatthecoresampledecontaminationprocedure inSubheading 3.2.2 andreimagethecoreuntilnomicrospheresare detected.
3.2.4GrindingofCore Samples
1.Performallworkinalaminarflowhooddedicatedtocore samples.
2.Wipeallsurfaces,equipment,andpipetteswith70%ethanol.
3.Putallreagents,supplies,andpipettesinthelaminarflow hood,UVsterilizefor30min.Rotatepipettesandsupplies, UVsterilizeforanadditional30min.
4.Spreadasheetofsterilealuminumfoilontheworkingsurface.
5.LightaBunsenburnerneartheopeningtothelaminarflow hood.Holdingthecoresampleinmetaltongs,quicklypassthe coresamplethroughtheflametwicewhilerotatingthecore slightly,andplaceonthealuminumfoilsheetinthelaminar flowhood.Allowthecoreexteriortocoolfor1min.
6.Usingasledge/drillinghammerandcoldchisel,breakthecore materialintosmallerpieces,untiltheywillfitinthePlattner’s mortarsleeve.Placethepestleinthemortarandstrikethe pestlewiththesledge/drillinghammer.Repeatuntilthesampleisbrokenintosmallerpieces(see Note5).
7.Oncethesampleisbrokenintosmallpieces(see Note6), transferthematerialtotheseriesofstackedsieves,withthe largestmeshatthetop.Gentlyshakewithaside-to-side motion.
8.Removethematerialfromthebaseofthestackedsievesand placeinasterileglassorplasticcontainerforstorage.
9.Removethematerialinthelargersievesthatdidnotpass throughtothebaseandplaceinthePlattner’smortarand pestleorastandardceramicmortarandpestletofurtherreduce thesize.Passthroughthestackedsievesuntilallsampleis groundtothedesiredsize.
10.Storegroundcoresamplesat 80 CuntilDNAextraction.
3.3DNAExtraction
Itisessentialtousealaminarflowhooddedicatedtocoresamples duringprocessing.Thoroughlycleanthehoodbeforeandafterany procedure.Theuseofextra-longnitrileglovestocovertheexposed wristregionanddisposableTyveklabcoatsisrecommended.Itis criticaltotreatevery“blank”exactlyastheactualsampleandevenif the“blanks”resultinnon-detectableDNA,tocarrythe“blanks” throughtolibrarypreparationandsubsequentsequencing. Low-levelcontaminationhasbeenwelldocumentedevenincommercialnucleicacidextractionandpurificationandamplification kits[21, 22].DNAsorptionontomineralsurfacesisanadditional problemwithlow-biomasssamples.Theuseofblockingagentsand carriermoleculeshavebeenshowntohelpovercomethisdifficulty [23, 24],andisextensivelypresentedin[16].Howeverourtests showedthatwiththeseshalecoresamples,blockingagentand
3.3.1DNAExtraction fromGroundCore
carriermoleculeshaddeleteriouseffectsonDNArecoveryand additionallycontainedhighlevelsofmicrobialcontamination.
1.Performallworkinalaminarflowhooddedicatedtocore samples.
2.Wipeallsurfaces,equipment,andpipetteswith70%ethanol.
3.Putallreagents,supplies,andpipettesinthelaminarflow hood,UVsterilizefor30min.Rotatepipettesandsupplies, UVsterilizeforanadditional30min.
4.Turnontheovento50 C.
5.Labelalltubes,includingthesamenumberoftubesforblanks asforeachsample(e.g.,ifyouareextractingfromatotal20g ofgroundsample,youwillneed401.5mLtubescontaining 0.5gofsampleeachand401.5mLtubesfortheextraction blank).
6.Sprayallsurfaceswith(first)DNAzapsolution1,thenDNAzapsolution2,wipewithsterilepapertowels,thenwithsterile water.
7.Put0.5ggroundshaleintoeach1.5mLtube(nothingfor blanks).Thetotalnumberof0.5galiquotsisdependentonthe totalmassofsampletoextractfrom(see Note7).
8.Add1.0mLoflysisbufferIsolutiontoeachtube,vortexto mixandfreezeat 80 C.
9.Thawsamples,vortex,andincubatefor1hat50 C,vortexing every10mins.
10.Centrifugesamplesat10k g for3minatroomtemperature, transfersupernatantsfromreplicateextractionstoa50mL conicaltube.
11.Put50mLtubeoniceandextractagainfromeachtube containinggroundshale,andextractionblanktubes,byadding 1.0mLoflysisbufferIsolution,vortextomix.
12.Centrifugesamplesat10k g for3minatroomtemperature, transfersupernatantsfromreplicateextractionstoa50mL conicaltube.
13.Addonevolumeofphenol:chloroform:isoamylalcoholtothe combinedsamples,mixbyinversion,centrifugeat10k g for 10minatroomtemperature,transfersupernatanttonew 50mLtube.
14.Addonevolumeofchloroform:isoamylalcoholtothesamples, mixbyinversion,centrifugeat10k g for5minatroom temperature,transfersupernatanttoanew50mLtube.
15.Repeatchloroform:isoamylpurificationin step14.
3.3.2Quantification ofDNA
16.Precipitatebyadding30 μLoflinearacrylamideand0.2 volumesof5MNaClsolution(see Note8).Calculatethe newtotalvolume,thenadd2.5volumesof100%ethanol. Theprecipitationmayneedtobedividedbetweenmultiple 50mLconicaltubes.
17.Incubatefor2hatroomtemperatureorovernightat4 C.
18.Centrifugesamplesat12k g for30minatroomtemperature.
19.Wash2 with70%ethanol,centrifugeatmaxspeedfor 5–15min,drypelletinthelaminarflowhoodbyinverting tubeonsterilealuminumfoil.
20.Resuspendpelletsin50–100 μLinwarm(50 C)EBbuffer. Usetheminimumvolumepossibletoresuspendpellets.
21.StoreextractedDNAat 80 Corquantifyimmediately.
Itisimportanttouseassmallavolumeofsampleaspossibleduring quantification.TheQubitdsDNAHS2.0fluorometerprotocol allowsforupto20 μLofsampletobequantified.Howeverthe sampleisnotrecoverableaftermeasurement.ItisnotalwaysnecessarytoquantifytheamountofDNArecoveredforextremely low-biomasssamples,asfuturelibrarypreparationformetagenomicsequencingcantolerateawidevarietyoftemplateconcentrations.TheQubitdsDNAHSfluorometer2.0assayhasa detectionlimitof10pg/μLwhenusing20 μLofsample.IfDNA concentrationsarebelowtheQubitdsDNAHSdetectionlimit, 10pg/μLcanbeusedasanupperlimittoestimatetemplateinput forlibrarypreparation.Otherfluorescence-basedassays,suchasthe Quant-iTPicoGreendsDNAassay,canalsobeusedbuttheuseofa nanodropisnotrecommended,assomeco-extractedcontaminants willcauseinterferenceandwillinflatethedetectedconcentrations.
1.Wipeallsurfaces,equipment,andpipetteswith70%ethanol.
2.Putallreagents,supplies,andpipettes(exceptforsamplesand Qubitstandards)inthelaminarflowhood,UVsterilizefor 30min.Rotatepipettesandsupplies,UVsterilizeforanadditional30min.
3.ThawDNAonice.
4.Prepareamastermixaccordingtothemanufacture’sinstructions,appropriateforthenumberofsamplestobequantified.
5.Aliquotsamplesandstandards.
6.MeasureusingtheQubitfluorometerandrecorddetected DNAconcentrations.
7.StoreextractedDNAat 80 Corproceedimmediatelyto metagenomiclibrarypreparation.
3.3.3LibraryPreparation andOptionalMDA
Amplification
TypicalmetagenomiclibrarypreparationsrequireinputDNAconcentrationsrangingfromseveralnanogramsuptoamicrogram.Yet DNAextractionsfromsampleswithlowcelldensityyieldDNA massesinthefemtogramtopicogramrange( 1fgDNApercell). MethodshavebeendevelopedtocircumventlowDNAyields,such asMDAamplificationwhichproducesmillionsofcopiesoftemplateDNAandisusedforsingle-cellgenomicamplification;commercialkitssuchastheNexteraXTDNAlibrarypreparationkit (recommendedinput1ngDNA),andtheAccel-NGS1SPlus librarykit(inputsaslowas10pg)whichwasdevelopedforancient, degradedand/orssDNA.DespitethefactthatMDAhasbeen showntohaveinherentbiasesduringamplification,itmaybe necessaryforcertainsampleswhenlibrarypreparationfailswith non-detectableinputDNA;alternatively,theextractedDNAcanbe splitandlibrariespreparedwithandwithoutMDAamplification. Itisessentialtousededicatedlaminarflowhoods.Thoroughly cleanthehoodbeforeandafteranyprocedure.Theuseofextralongnitrileglovestocovertheexposedwristregionanddisposable Tyveklabcoatsisrecommended.Itiscriticaltotreatevery“blank” exactlyastheactualsampleandevenifthe“blanks”resultin non-detectableDNA,tocarrythe“blanks”throughallanalyses. Extremediligenceisrequiredinordertopreventcontaminationof samples,andinadvertentamplificationofcontaminatingDNA. ContaminationofasinglecellorDNAfragmentwilldistortdownstreamanalyses.
1.Carefullyreadthemanufacturerprotocolsandrecommendationstoensurelibrariesarerepresentativeoftheoriginal sample.
2.Performallworkinalaminarflowhooddedicatedtocore samples.
3.Wipeallsurfaces,equipment,andpipetteswith70%ethanol.
4.Putallreagents,supplies,andpipettesinthelaminarflow hood,UVsterilizefor30min.Rotatepipettesandsupplies, UVsterilizeforanadditional30min.
5.Sprayallsurfaceswith(first)DNAzapsolution1,thenDNAzapsolution2,wipewithsterilepapertowels,thenwithsterile water.
6.ForoptionalMDAamplification,followthemanufacturerprotocolwiththeserecommendations:
(a)IfperformingMDAamplification,besuretoincludea no-templatecontrol.
(b)UsetheQiagen“WholegenomeamplificationfromgenomicDNAusingtheREPLI-gSingleCellKitwith increasedsamplevolumes.”Thisincreasesthevolumeof templatetoaddfrom2.5to15 μL.
(c)Anincubationtimeoflessthantherecommended8hmay yieldbetterresults.Itmaybeusefultosetupreactions thatincubatefor4,6,and8h,andcarryallthroughto sequencing.
(d)QuantifyMDA-amplifiedDNAusingtheQubitprotocol inSubheading 3.3.2.AmplifiedDNAmayneedtobe diluted1:100or1:1000tobeinthequantificationrange.
7.FortheNexteraXTDNAlibrarypreparationkit,followthe manufacturerprotocolwiththeserecommendations:
(a)DonotoverdrytheAMPureXPmagneticbeadsorthe DNAwillnotberecoverable.
(b)Checkthequalityandsizeoflibrarypreparationona BioanalyzerHighSensitivityDNAchip.
(c)Contactyoursequencingfacilitytodetermineifyou shouldproceedthroughtheNormalizeLibrariesstep. Manysequencingfacilitiesprefertoperformthisstep.
4Notes
1.Thedrillingprocesscantakedaysorweeks.Itisadvantageous totakesamplescontinuouslyduringthedrillingprocess,especiallywhennewfluidsandchemicalsareaddedtothe drillingmuds.
2.ExtractionofDNAfromdrillingmudsusingthemethod describedforcoresamplescanresultinviscous,unusable DNA,duetocoextractionofdrillingmudcomponents.We obtainedthebestresultsusingtheMoBioPowerMaxSoil DNAisolationkitfordrillingmuds.RegardlessoftheDNA extractionmethodused,nucleicacidsareoftenstillcontaminatedwithsubstancesthatinhibitPCRreactionsormetagenomicsequencingduetothecomplexnatureofthesamples. Wehavefoundthatasecondarycleanupofthenucleicacidsis usuallyrequired.TheGenomicDNAClean&Concentrator10kitfromZymoResearchgivesgoodresults,althoughmany othercommercialkitsareavailable.Ethanolprecipitationcan haveadverseeffects,resultingintheconcentrationofnucleic acidsaswellasinhibitorysubstances.
3.Communicationwiththeoperatoron-siteiscriticaltoobtain representativesamples.Wellinadvanceofsampling,meetwith theoperatorstodeterminetheamountofsiteaccessyou’ll have,andwhomtocontactduringeachphaseoftheoperation. Providealistofsamplesneededandinstructionsforsterile techniqueiftheoperatorswilltakethesamples.
4.Bentonite,aclayaddedtodrillingmudsinordertoprotectthe formationfrominvasionofdrillingfluids,hasautofluorescent properties.Besuretotestvisualizationofmicrosphereswitha sampleofdrillmud,beforeaddition.Toomuchbentonite coatingcorescaninterferewithvisualizationoffluorescent microspheres.
5.BecarefulnottostrikethePlattner’spestlewhenthesleeveis crookedonthebase,orcoreparticlesarebetweenthesleeve andthebase.Testbyplacingthepestleinthesleeveandfirst rotatingthesleeveonthebase.Nextrotatethepestleinthe sleeve.Ifthesleeveandpestlerotatesmoothly,thepestleis seatedproperlyandcanbestruckwiththesledge/drilling hammer.Failuretoproperlyseatthepestlecanresultinsample lossandabentsleeve,makingthePlattner’spestleunusable.
6.ThedesiredsizeofcorematerialfromthePlattner’smortarand pestledependsonthehardnessandporosityofeachsample type.Forsomesampletypes,itmaybeeasiertofirstreducethe sizeofthecorematerialinthePlattner’smortarandpestle, thentransferthematerialtoastandardceramicmortarand pestle,usingacircularmotiontoreducethesizefurtherbefore sieving.Themeshsizeofeachsievecanbemodifiedbasedon thesampletype,resultinginlargerorsmallerparticlesas desired.
7.Theamountofsampletoextractfromisdependentonthe samplematrixandtheamountofbiomass.Itmaybenecessary toextractfrom50–100gofsampletoobtainenoughDNAfor sequencingpurposes.
8.ThefinalconcentrationofNaClduringtheprecipitationprocessislowerthanrecommendedformostprotocols,including theprotocolherethatweadaptedfrom[16].Weoptimizedthe concentrationofNaClforourparticularsamples,asshalecontainshighamountsofsaltsduetothefactthattheyareusually originallydepositedasmarinesediments.TestsofprecipitationsusingrecommendedNaClconcentrationsresultedin recoveryofalargesaltpelletwithlittletonoquantifiable DNArecovered.Thisillustratestheimportanceoftestingall protocolsbeforeperformingthemonactualsamples.
Acknowledgments
RebeccaDaly,KellyWrightonandMichaelWilkinswerepartially supportedbyfundingfromtheNationalSciencesFoundation DimensionsofBiodiversity(AwardNo.1342701)andbythe MarcellusShaleEnergyandEnvironmentLaboratory(MSEEL) fundedbyDepartmentofEnergy’sNationalEnergyTechnology laboratory(DOE-NETL)grantDE#FE0024297.
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FreshwaterViromes:FromSamplingtoEvaluation
CatherinePutonti,Zoe ¨ Diener,andSiobhanC.Watkins
Abstract
Thereareanumberofoptionsavailabletoresearcherswhowishtocollectandanalyzeviralmetagenomes (viromes)fromenvironmentalsamples.Herewedescribealaboratoryprocedureforgenerationofviromes fromfreshwatersamples,specificallytargetingdsDNAbacteriophages.Wealsodiscussmethodsforbioinformaticanalysisoftheresultingdata.
Keywords Viromes,Metagenomics,Freshwaterviruses,Bacteriophage,Viralbioinformatics
1Introduction
Virusesevolverapidly,demonstrateveryhighlevelsofgenomic plasticity,andareanunderstudiedgroupincomparisontoprokaryotes.Whilemetagenomicsapproacheshavefilledinthe“missing branches”oftheprokaryotictreeoflife[1],sequencingofcomplex viralcommunitieshastodateonlysampledafractionofthediversitylikelypresent(e.g.,[2]).Manyofthesequencespresentina givenviralmetagenome(henceforthreferredtoasavirome)dataset willhavenoknownhomologsintheexistingsequencedatabases. Insomeenvironments,thenovelfractioncanbeupwardof90% [3–5].Inaddition,phylogenyispoorlytraceableowingtothelack ofauniversalgenemarkerequivalenttothe16SrRNAgene. However,someviruses,particularlybacterialviruses(bacteriophages),arewellsuitedtometagenomics-basedanalysis,dueto theiraccessibilityandabundanceincertainsampletypes.
Preparationofvirusesforviromeanalysismayvarywithsample, orbythequestionbeingasked,butforwater,allobserveageneral workflow:filtrationandconcentrationoftheviralfraction,preparationandsequencingofDNA,andDNAsequenceanalysis.Concentrationmaybeachievedphysically,viatangentialflowfiltration (TFF)orultracentrifugation,orviachemicalflocculation(e.g., FeCl3).However,purificationofadequatequantitiesofDNA usingthesemethodsmaystillnotbepossible.Insuchcases,
RobertG.Beikoetal.(eds.), MicrobiomeAnalysis:MethodsandProtocols,MethodsinMolecularBiology,vol.1849, https://doi.org/10.1007/978-1-4939-8728-3_2, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2018
amplificationprotocolscanbeusedtobolstertheDNAfoundin thesample—however,theseareassociatedwithinherentandconsistentbiases[6].Currentmethodologyforassessmentofviromic datasetsreliesheavilyonhomology-basedcomparisonsto sequencedandcharacterizedviralgenomes.Onlyasmallfraction oftheextantviraldiversity—estimatedlessthan1%—hasbeen characterized[3].Ofthesequencedvirusescurrentlyavailable, thereisalsoaclearbias:virusesthatinfecthumansorcanbeeasily propagatedwithinthelabareoverrepresented[5].Bioinformatic analysisisfurthercomplicatedbytheinherentchallengesposedby geneticmobility[7].
Thismethodoutlinesabasicprotocolforpreparationand analysisofaviromegeneratedfromfreshwatersamples,targeting dsDNAbacteriophages.WhileRNAandssDNAphagesareimportantmembersofviralcommunities[8],theseviralspeciesnecessitaterefinedmethodsforsampling,genomicextraction,and amplification[5].Assuch,thediversityofRNAandssDNAphages isseverelyundersampled,althoughafewfreshwaterviralcommunitieshavebeensurveyed(e.g.,[9–12]).Here,wealsodescribe bioinformaticapproachesfordataassessment.Thisisparticularly importantforfreshwatersandsamplesofasimilarmicrobiological complexity,whicharelikelytocontainalargefractionofpreviously undiscoveredgeneticdiversity.
2Materials
2.1SamplePrep, NucleicAcid Extraction, SequencingPrep
1.Sterilecontainer(e.g.,bottle,carboy)tocollectwatersample. See Note1 withrespecttovolumeforsampling.
2.100mL0.45 μmMicroFunnel™ DisposableFilterFunnels (cat.No.4804;PallLaboratory,PortWashington,NY)[QiagenDNeasy® PowerWater® Kitrecommendation]or500mL sterilebottle-topfilters,0.45 μmcelluloseacetatemembrane filters(cat.No.430514,CorningInc.,Corning,NY).
3.100mL0.22 μmMicroFunnel™ DisposableFilterFunnels (cat.No.4803;PallLaboratory,PortWashington,NY)[QiagenDNeasy® PowerWater® Kitrecommendation]or500mL sterilebottle-topfilters,0.22 μmcelluloseacetatemembrane filters(cat.No.430513,CorningInc.,Corning,NY).
4.Chloroform,biotechnologygrade(cat.No.VWRV0757500ML,VWR,Radnor,PA).
5.OPTIZYME™ DNaseI(RNase-free)(cat.No.BP81071, FisherBioreagents,Pittsburg,PA).
6.RNAseOne™ (cat.No.M4261,Promega,Fitchburg,WI).
7.Labscaletangentialflowfiltration(TFF)system(cat. No.XX52LSS11,EMDMilliporeCorp,Billerica,MA).
2.2Sequencingwith IlluminaMiSeq
2.3SequencingData QualityControland Assembly
2.4Virome ClassificationTools
FreshwaterViromes:FromSamplingtoEvaluation19
8.PelliconXLFilterModuleDurapore0.1 μm50cm2 polypropylenefilter(cat.No.PXVVPPC50,EMDMilliporeCorp, Billerica,MA).
9.Sterile15mLcentrifugetube.
10.QiagenDNeasy® PowerWater® Kit(cat.No.14900-50-NF, Qiagen,Germantown,MD).
1.Qubit™ 4Fluorometer(cat.No.Q33226,Invitrogen,Carlsbad,CA).
2.Qubit™ dsDNABRAssayKit(cat.No.Q32850,Invitrogen, Carlsbad,CA).
3.QubitAssayTubes(cat.No.Q32856,Invitrogen,Carlsbad, CA).
4.NexteraXTDNALibraryPreparationKit(cat. No.FC-131-1024,Illumina,SanDiego,CA).
5.MiSeqReagentKitv2(500-cycles)(cat.No.MS-102-2003, Illumina,SanDiego,CA).
1.ComputerwithaUNIX-basedoperatingsystemandatleast 8GBofRAM.
2.Software:Sickle[13]version1.33orlater.
3.Software:FastQC[14]version0.11.7orlater.
4.Software:SPAdes[15]version3.9.0orlater.
1.ComputerwithaUNIX-basedoperatingsystem(ifperforming genepredictions,otherwiseanyoperatingsystemcanbeused) andatleast4GBofRAM.
2.Blast+Executable[16].
3.GeneMarkS[17]v.4.30orlater.
4.InformativityAnalysisTool[18]v.1.0.
3Methods
3.1SamplePrep, NucleicAcid Extraction, SequencingPrep
1.Collectviralparticlesfromfreshwatersamplesbysequential filtrationandconcentration.Passwaterthroughsterile 0.45 μmand0.22 μmfilterstoremoveeukaryoticcellsand debrisandprokaryoticcells,respectively.
2.Filterandconcentrateusinga0.10 μmfilterattachedtoaTFF system,collectingthefinalfiltrateinasterile15mLcentrifuge tube.FollowthefiltrationprocessoutlinedintheLabscaleTFF protocol.Thisfinalconcentration(3–10mLinfinalvolume) containstheviralfractionofthesample,fromwhichDNAwill beextracted(see Note2).
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300.000 nummi (E . o. c. 11). Cfr. su questo anche M , in Hermes, 1890, p. 27.
520. E . o. c. 6.
521. C , Le Conseil des empereurs, 473.
522. Lo notarono anche i contemporanei; cfr. E . o. c. 5 I . C . De vita monast. 3, 5.
523. Cfr. le due lettere di Ottaziano Porfirio e di Costantino in O . P . Carmina, ed. M , pp. 3, 4.
524. A . V . Epit. 41, 8.
525. I . Orat. 1, 8 c., ed. H .
526. A . V . Epit. 41, 14.
527. C . Antiqu. const. P. 42 d. A . Ant. const. 1, 31 (in B , o. c. I, 3, p. 12). Veramente; l’uno e l’altro dicono soltanto che la successione dei maestri, adibitivi all’insegnamento, durò 414 anni, fino al 10º dell’impero di Leone Isaurico. La fondazione sarebbe dunque avvenuta nel 313 di C., innanzi cioè quella di Costantinopoli.
528. G , o. c. III, 30 C . Griech. Litter. 809, (4ª ed.)
B , Κωνσταντινόπολις, Atene, 1890, I, 458.
529. A . o. c. I, 31 C . o. c. P. 42 d. Z . 15, 3, 13-16 (= P. II. 104 b. c.) C . P. 454 c. d.
530. Cfr. M , Ueber das rhetorische Gepräge d. römischen Litteratur, in Vermischte Abhandlungen u. Aufsätze, Breslau, 1821, p. 83, n. x.
531. Notitia urbis constantinopolitanae, 9, 10 (in Notitia dignitat. ed. S ). Cod. th. 14, 9, 3. Sulla Basilica Capitolina, cfr. Anth. pal. 9, 660 B , o. c. II, 853 B , o. c. I, 283.
532. A . Enarr. Chronogr. antiqu. Constant. 296 (in B o. c. I, 103).
533. Cfr. H , Die Geschichte d. Griechenland unter d. Herrschaft. d. Römer, Halle, 1875, III, 494. G , Geschichte d. öströmischen Reiches unter den Kaisern Arcadius u. Theodosius II., Halle, 1885, p 275
534. S . H. E. 3, 1 b.
535. Cfr. il cap. VIII del pres. scritto.
536. S . l. c.
537. Anth. pal. 9, 660.
538. H . Chronicon (ed. S , II, 195) A . XVI, 2, 4 T . Or. 23, p. 292 a sgg. Cfr. M , De rhetoricae discipulis atque magistris per Orientem in IV. Cristiani aevi saeculo, Parisiis, 1866, pp. 41-42.
539. Cod. th. 14, 9, 3. 6, 21, 1.
540. Cod. th. 6, 21, 1.
541. Curiosum Urbis e Notitia (in R , Topographie d. Stadt Rom, p. 375)
542. T . Orat. 23, p. 294 b.
543. S . Malchus.
544. Cod. th. 13, 4, 1.
545. Se noi fossimo sicuri che non ci sia errore materiale in una delle nostre fonti, potremmo anche discorrere di una vera e propria biblioteca di libri di meccanica raccolta da Costantino in un apposito edificio, i Μάγγανα, ch’era altresì un arsenale di macchine e di materiali di guerra (G . Ann. 3, P. 252 o un codice de l’A . Antiqu. constant. 2, 69 in B , o. c. II, 606). Ma è lecito sospettare che il testo originario, in luogo di βίβλοι μηχανικαὶ, abbia discorso di ὕλαι μηχανικαὶ (cfr. B , l. c.).
546. Cfr. Cod. th. 11, 27, 1-2.
547. Cod. th. 13, 3, 1.
548. Cfr. il commento del G alla legge.
549. Cod. th. 13, 3, 2.
550. Cod. th. 13, 3, 3.
551. Cfr. P . V. S. 2, 25, 5 e G (V, 29) nel suo commento al Cod. th. 13, 3, 1. K , Historia originis ac progressus scholarum inter Christianos, Helmstadi, 1743, pp 41 sgg
552. Cod. th. 13, 3, 16.
553. Di tale fatto ci fornirebbe un’indiscutibile riprova una variante, che del passo della legge ci è offerta in alcuni mss. del C. I. 10, 53, 6, dove essa verrà riprodotta e dove, insieme con gli altri beneficati, si elencano i doctores legum, se però quella variante potesse sicuramente interpretarsi come una meditata interpolazione dei giurecousulti compilatori del C I Cfr D , Die Institutionen d Caius, Halle, 1869, p 8, n 14
554 quo facilius liberalibus studiis et memoratis artibus multos instituant
555 Cod th 13, 4, 2 (= C I 10, 66, 1)
556 Il Cod th ha albarii; il C I , albini e dealbatores
557 I codici hanno medici, ma l’inclusione dei medici tra questi professionisti non si spiega, e deve trattarsi di un errore
558 Per indicare gli indoratori, il testo adopera barbaricarii e deauratores Sulla differenza di significato tra questi due sinonimi, cfr B , Adnot ad Notit dignitat , Bonnae, 1839-1853, II, 1, pp 364-365
559 Il C I (l c ) aggiunge: gli scavatori di pozzi (o lectarii, fabbricatori di letti?), i magnani, i cocchieri (o costruttori di quadrighe?), i fabri (meccanici?), i sarti, i piumai (o ricamatori?), i coniatori, i lavoratori di lino Ma sul valore e la paternità di queste aggiunte del C I , data la grande libertà e varietà di criteri, cui si attennero i compilatori, non possiamo dire nulla di sicuro
560 CIL 6, 1708 — Notitiadignit occid 4, 14; cfr H , o c p 272 — D R , Diz. ep. II, 1327.
561 A M 16, 6, 2; cfr W , Gesch d Kunst, in Werke, Donaueschingen, 1825, VI, 346-348
562. Cfr. B , Grundriss der griech. Literatur, I4 , 656.
563. E . V. S. p. 492, ed. B .
564. Fragm. vat. 150, ed. M .
565. Cod. th. 13, 4, 3 (= C. I. 10, 66, 2) e commento del G .
566. T . Or. 4, 59 d. sgg. Non si tratta di una nuova pubblica biblioteca, come pure è stato creduto. Il passo di T non autorizza in nessun modo a ritenerlo, e il Cod. th. 14, 9, 2, come S . Malchus, parlano di una sola pubblica biblioteca costantinopolitana
567. L . Or. 1, p. 27, ed. R .
568. L . Or. 1, pp. 52-54; 58; 126.
569. L . Or. 1, p. 27.
570. L . Or. 1, p. 36.
571. L . Or. 1, p. 52 sgg.
572. L . Or. 1, p. 58.
573. E . V. S. p. 487 P I , L’école d’Athènes, Paris, 1868, pp. 29; 33-34.
574. C . Oratio ad Them. (in T . Orationes, ed. D , p. 21 sgg.) pp. 20 a-21 c.
575. T . Or. 4, 61 a-b.; cfr. anche p. 54 d.
576. Cod. th. 13, 3, 4.
577. Cod. th. 12, 1, 36; 41; 42; 44.
578. Cfr. il commento del G a Cod. th. 13, 3, 4.
579 Cod th 13, 3, 2; cfr 6, 21, 1
580 Cod th 13, 3, 4; cfr il commento del G — I Ep 26, ed. H .
581 Cfr pp 236-237 del pres scritto
582 K o c I, 89 I Ep 45
583 Z 3, 11, 3
584 I Epist ad S P Q Athen p 277 c
585. I . Ep. 9; 36.
586. Cod. th. 13, 3, 5. Nel C. I. 10, 53, 7, che la riproduce, manca, grazie alla libertà dei compilatori, l’ultima parte, relativa all’autorizzazione del principe.
587. N , Giuliano l’Apostata, Milano, 1902, 2ª ed., p. 327 A , Julien l’Apostat, Paris, 1903; II, 354 e passim, e così la maggior parte degli storici. Fra le poche eccezioni parmi debbano annoverarsi il M , Flavius Claudius Julianus, Gotha, 1867-1869, 2, 81 sgg e il G nel suo commento
588 Il R , Gesch d Reaction Kaiser Iulianus etc , Jena, 1877, p 64
L , Della politica religiosa di G imperatore, Piacenza, 1887, pp 110-111 N , o c p 329
589 Cod th 13, 3, 6
590 M , o c 2, 81
591 I Ep 42; cfr anche A M 22, 10, 7-25, 4, 20
592. Questa mi pare la più ragionevole interpretazione di questo passo, che vedo invece reso da altri diversamente: πῶς οὐ τοῦτο
593 G N 4, 5-6; 101 sgg A De civ Dei, 18, 52 R H E 10, 33, ed M S H E 3, 16 c S 5, 18 b
594 Cfr su ciò anche R , o c 66, n 8
595 κακουργία (In Iuvent et Maxim 1)
596 S H E 3, 16
597. N o. c. p. 335.
598. E . V. S. p. 482, ed. B :
599. Or. 18, p. 574.
600. D B , L’Église et l’empire romain au IV. siècle, Paris, 1862, IV, pp. 209-210; 213; 217.
601. A , o. c. II, 357 agg.
602. H -K , Storia universale della Chiesa, (trad. it.), Firenze, 1904, I, 14.
603. D B , o. c. IV, 217.
604. L , o. c. 110-111.
605. G , The history of decline and fall of the roman Empire, 1829, IV, pp. 92 sgg. B , Zur Beurteilung d. Kaisers Iulianus, Bayreuth, 1891 (progr.) 23-24. È questa la tesi ampiamente svolta da S. Gregorio di Nazianzo (Or. 4, 5, sgg.), il teologo di quel tempo più violento contro Giuliano. Ma (singolare contradizione!) le due orazioni di S Gregario contro Giuliano sono per buona parte un attacco vivacissimo contro la cultura classica e la immoralitè dell’insegnamento, che è possibile ritrarre dagli scrittori pagani
606 D M , La libertà di riunione; di associaz etc in Rendiconti dell’Istituto lombardo di sc e lett , 1900, p 851
607 Ad es il M , Études morales, Paris, 1883, p 294
608 M , o c 2, 84
609. N , Julian l’Apostat et sa philosophie du polythéisme, Paris, 1877, pp 170-172
610. G , Iulian philosopher and Emperor, New-York, 1895, p. 239-240.
611. N , o. c. 344 sgg.
612. P . Protag. 15.
613. Sui criteri pedagogici, informatori delle scuole di retorica, cfr. B , Fin du paganisme I, 218 sgg. e le acute osservazioni, di cui è cosparso uno scritto, che gli storici di solito non leggono, S , La ruine du monde antique, Paris, 1901, pp. 69 sgg.
614. Per le scuole famose di Port-Royal, cfr. C , Les pédagogues de Port-Royal, Paris, 1887, pp. XVII-XVIII; 60-61; 61, nn. 1 e 2; 272 sgg.
615. Si potrebbe dire di più: il passo dell’editto di Giuliano (Ep. 42 c.), che richiedeva che i maestri non nudrissero opinioni contrarie a quelle da loro professate in pubblico (μὴ μαχόμενα
φέρεν δοξάσματα) è stato con cecità partigiana, anche dai migliori (cfr A , o c II, 357), interpretato come recante l’imposizione di una conformità di vedute tra i maestri e l’opinione pubblica Tale interpretazione, se stenta ad accordarsi con la grammatica, termina certamente per attribuire a Giuliano il più illogico e il più sbagliato dei ragionamenti
616 N , o c 344 sgg
617 Cfr i Cap VIII e IX del pres scritto
618 S H E 3, 16
619 È stato da più di un moderno ricordato che, anche ai nostri giorni, degli ecclesiastici hanno chiesto il bando degli autori classici dalle scuole (B , o c I, 353) Ma essi non hanno rilevata la singolare, ma non istrana, coincidenza, per cui le scuole cattoliche, che sono tutte confessionali, e il cui grande pregio è di inculcare una fede, e di farne il fuoco centrale ispiratore dell’educazione e dell’insegnamento, ripetono, con le opportune, o necessarie, mutazioni di mezzi e di fini, la loro natura dal criterio fondamentale dell’editto di Giuliano.
620. D B , o. c. IV, 213.
621. A . Confess. 8, 5, 10.
622 E V S p 492
623 I Ep 2
624 H Chron ad a 366 (II, 196 ed S )
625 H l c E V S p 493 Non ho potuto vedere il L , Influence des Pères de l’Église sur l’éducation, ove, secondo trovo riferito, si sostiene che Proeresio non sarebbe stato cristiano
626 O 7, 30, 3 I C In Iuv et Maxim 1
627 M , o c 2, 82 B , o c 22
628 D B , o c IV, 216 e n 1 G , Decline and fall of the rom empire, IV, 93 L , Der Untergang d Hellenismus, München,
1854, p. 64, n. 184 R , o. c. 66 A , o. c. II, 363-364.
629. S . H. E. 2, 46, 3, 16 a S . H. E. 5, 18 c.
630. G . N . Orat. 4, 111-112; cfr. S . H. E. 5, 16.
631. 14, 6, 18.
632. Cfr. anche H , Les écoles d’Antioche, Paris, 1898, 114 sgg.
633. pari a ca. l. 64. Sull’arruffata questione della capacità dell’artaba, nell’età imperiale romana, cfr. H , Beiträge zur Aegyptischen Metrologie, in Archiv f. Papyrusforschung etc. II, 283 sgg. G -H , in Tebt. Pap. I, 232-233 B , Contributo alla storia economica dell’antichità, Roma, 1907, pp. 57-59.
634. Ep. 56.
635. Ep. 71.
636. Com’è noto, l’autorità delle lettere di Giamblico a Giuliano è stata più volte posta in dubbio (S , De vita et scriptis Iuliani imperatoris, Bonn, 1888, pp. 23 sgg. Z , o. c. III4 , 2, 736-8, n. 3); ma quei dubbi non hanno in verità fondamenta troppo solide (C , Hist de la litter grecque, Paris 1899, V, 888 e n 1 N , o c 451, n 1)
637 Ep 3
638. Ep. 40.
639. Ep. 4.
640. Ep. 15.
641. 22, 7, 3, cfr. L . Or. 18, p. 574.
642. A . M . 25, 3, 15 sgg.
643. Cod. th. 13, 3, 6.
644. Così mutilata la ritroviamo nel C. I. 10, 53, 7.
645. A . M . 23, 5, 11.
646. Cod. th. 14, 9, 1.
647. Queste consociationes debbono essere state le corporazioni degli studenti, i cui atti talora criminosi sono più volte censurati dagli scrittori contemporanei
648. I corporati erano persone, facenti parte di associazioni speciali, riconosciute dallo Stato, le quali, nel IV e nel V secolo di C , ebbero una importanza massima nella vita dell’impero, segnatamente in Roma e in Costantinopoli, e vennero incaricate di speciali servizi pubblici, in cambio dei quali godevano determinati privilegi; cfr W , Les corporations professionelles chez les Romains, Louvain, 1896, II, 139 sgg ; 193 sgg e passim
649 Cfr , oltre a quello del G , il bel commento alla legge del C , in D S , Novus thesaurus antiquitatum, III, Venetiis, 1735, pp 1199-1232, nonchè le osservazioni del K , o c § 12-16 e del V , Essai sur l’histoire de la praefectura urbis à Rome, Paris, 1896, pp. 305; 118.
650. Le fonti sono Libanio, S. Gregorio di Nazianzo, S. Agostino. Per un quadro generale di quella vita e di quell’ambiente, cfr. H , o. c. III, 349 sgg. H , Les écoles d’Antioches, pp. 205 sgg. M , Les Africains, Paris, 1894, 66 sgg. R , o. c. 29.
651. A . Confess. 5, 8, 14: quietius studere adulescentes et ordinatiore disciplinae coercitione sedari.
652. Cod. th. 13, 3, 10.
653. Cod. th. 13, 4, 4. Il testo dà picturae professores. Tale epiteto non basterebbe a designare dei maestri. Ma la legge è richiamata in un’altra di Teodosio II. (Cod. th. 13, 3, 18; cfr. C. I. 12, 40, 8), rubricata sotto il titolo de professoribus, che questa volta sono realmente insegnanti pubblici e privati.
654. Era un’imposta che gravava sui mercanti.
655. A . M . 30, 9, 4.
656. M , Röm. Strafrecht, Leipzig, 1899, 249-250.
657. Questa è la più probabile interpretazione della seconda tra le clausole da noi enumerate della legge di Valentiniano. Essa dette luogo a un’interessante discussione tra il S (Römische Steuerverfassung unter d. Kaisern in Verm. Schriften, II, 83-84) e lo Z
L (Zur Gesch. d. röm. Steuerwesen in d. Kaiserzeit, estr. dalle Mémoires de l’Académie imper. des sciences de S. Pétersbourg, 1863, pp 5 sgg) Cfr anche P , o c 95 sgg
658. Cod. th. 13. 4, 1; 2.
659. Orat. 9, p. 123 b.
660. Cod. th. 14, 9, 2.
661. Era questa la forma di rimunerazione, adottata ora anche per i pubblici docenti; cfr., ad es., T . Or. 23, p. 292 a sgg.
662. C , in D R , Diz. ep. III, 282 sgg.
663. Z . 4, 14-15.
664. A . M . 29, 1, 41. Perchè, ad es., i libri di diritto?
665. Cfr. anche S . H. E. 6, 35 e B , Ueber die Chronik d. Sulpicius Severus in Gesammelte Abhandlungen, Berlin, 1885, II, 102.
666. Orat. 10, p. 129 d-130 a.
667. Cod. th. 13, 3, 11.
668. Questa singolare modestia di stipendio del grammatico greco di Treviri si può spiegare col fatto che, in questa città, l’uso del greco era raro, l’apprendimento svogliato (cfr. A . 16, 9 ed. S ) e l’insegnamento, quindi, negletto come cosa superflua.
669. B , Die Diokletianische Taxordnung vom Jahre 301, in Zeitschrift für die gesammte Staatswissenschaft. 1894, p. 197 M , Die wirtschaftl. Entwickelung etc. in Jahrb. f. N. Ö. 1895, p. 742 e nota.
670. Leges novellae ad Theodosianum pertin.; Val. 13, 3, ed M M ; cfr. B , in Vierteljahrschrift für Social u. Wirtschaftsgesch. 1906, pp. 659 sgg.
671. I, 27, 1, 22 sgg.
672. CIL. 3 suppl. 2, p. 235828 , l. 1 a.
673. G . L . p. 34, ed. F .
674. T . Or. 23, p. 292 a.
675. Edict. de pretiis etc. 3, 3, ed M -B .
676. Secondo l’Edictum de pretiis (2, 1 sgg.), il vino, nell’impero romano, era uno dei prodotti più costosi.
677. Cfr. C , in D R , Diz. ep. III, 285 sgg.
678. 16, 9, 6: fructus exilis tennisque sermo.
679. Cod. th. 13, 3, 8 (= C. I. 10, 53, 9); 9 S . Ep. 10, 27 (= 10, 40= 10, 47) 2 sgg. V , in Revue arch. 1880 (39) p. 355 sgg.
680. Cod. th. 13, 3, 10; 12.
681. Cod. th. 15, 1, 14. Cfr. A . M . 27, 3, 10.
682. Cod. th. 15, 1, 19.
683. A . Gratian act. 17 e passim. Su Ausonio precettore di Graziano, cfr. I , Ausone et son temps, in Revue histor. 1891 (47) 256 sgg.
684. C , Hist. de la litt. grecque, V, p. 863.
685. B , Fin du paganisme, II, 209-210.
686 Così suona il lamento degli Ellenofili; cfr L Orat 1, p 133
687 B , o c II, 437
688 T Or 5, p 63 c; 9, p 123
689 Sulla reazione religiosa di Teodosio, cfr L Der Untergang d Hellenismus, München, 1854, pp 98 sgg S , Gesch d Untergangs d Heidentums, Jena, 1887, I, 257 sgg
690 S , o c I, 259; 276 sgg e fonti ivi cit
691 Cod th 12, 1, 98 (= C I 10, 32, 35)
692 Cod th 12, 1, 86; 87; 90; 91; 93; 94
693 Cod th 12, 1, 98: ne quid patriae periisse videatur
694. S . 5, 35 (= 33). Noi conosciamo il destinatario solo attraverso le poche lettere indirizzategli da Simmaco, che vanno dal 382 al 389. D’altra parte, fino al 380, il diritto a cotali stipendii non era stato messo in discussione (cfr S 1, 79 (= 73) )
695 Cfr P J , L’école d’Athènes, p 128
696. Cod. th. 13, 3, 13; 14; 15.
697. Cod. th. 16, 10, 8.
698. Ad es. il M , o. c. p. 47.
699. Ep. 10, 27 (= 40 = 47).
700. Il G e, sulla sua fede, anche lo S (o. c. I, 256 e n. 1) pensa sia stato un tempio della metropoli di quella regione, Edessa, quello stesso, che Libanio celebra nella sua orazione Pro templis, 10. Il D (Hist. ancienne de l’Église, Paris, 1906-07, II, 631, n. 2) pensa che si tratti invece della città di Harran, l’antica Charrae.
701. A . M . 14, 6, 12 sgg. Ammiano compose le sue Istorie verso il 390; cfr T , Gesch d röm Litt , II5 , 1093
702 Intendi le biblioteche private, come il testo chiarisce
703 A M 14, 6, 18-19
704. Cod. th. 15, 1, 37 (= C. I. 8, 11, 13) C. I. 1, 24, 1.
705. I due editti sono indirizzati a un Teodosio, allora praefectus praetorio delle Gallie (C . 17, vv. 50 sgg.).
706. Cod. th. 16, 10, 15; 18 (= C. I. 1, 11, 3). Circa i paesi, cui il primo si riferirà, cfr. il commento del G .
707. Cod. th. 16, 10, 16.
708. E , Histoire générale de la littérature du Moyen âge en Occident (trad. fr.) Paris, 1883, I, 298.
709. Advers. Symmachum. I, vv. 501-505.
710. G , Atenaide, storia di un’imperatrice bisantina (trad. it.), Torino, 1882, pp. 47 sgg.; 55 sgg. G , Geschichte d.
öström. Reiches, , 223.
711. Cod. th. 13, 3, 16 (= C. I. 10, 53, 11); 17.
712. Cod. th. 15, 1, 53. 14, 9, 3 (= C. I. 11, 19, 1-2). 6. 21, 1 (= C. I. 12, 15, 1).
713. Ciò è detto implicitamente nella legge del marzo (Cod. th. 6, 21, 1 C. I. 12, 15, 1). Per la emendazione dei passi corrotti di questo testo, ho seguito le ipotesi del G , accolte anche dal M , nella sua edizione del Codex Theodosianus.
714. H , Gesch. Griechenlands unter d. Herrschaft d. Römer, III, 272 G , o. c. 275 B , A history of later roman Empire from Arcadius to Irene, London, 1889, I, 128. Il G (o c 120-121) oscilla fra le due opinioni
715 S , Hist de l’école d’Alexandrie, Paris, 1845, II, 371 sgg V , Hist critique de l’école d’Alexandrie, Paris, 1846, II, 192 sgg — P J , o c pp 129 sgg — Z , o c III4 , 2, 805 sgg.
716 G , o c 278
717 G T Mirac 1 praef E CDXXXVIII, 10 (p 301302), ed V ; cfr G , o c 277-278
718 Si desume, confrontando il Cod th 6, 21, 1 con il Cod th 14, 9, 3
719 Tale infatti fu Elladio, un dotto, che aveva risieduto in Alessandria fino al 381, ove, per giunta, era stato sacerdote di Giove (S H E 5, 16 a)
720 H , o c 234-235; 240 sgg e fonti ivi cit
721 Cfr P , Die Regionen d Stadt Rom, p 170, n *
722 Cfr S , Storia del diritto rom nel M E (trad it ), Torino, 1854, I, 262, n c ; 263, n i
723 CIL 6, 9858, illustrata in Boll crist 1863, p 14 Sul retore privilegiato nel VI secolo, cfr J , in Berichte über die Verhandlungen d Königlich-Sächsischen Gesellschaft d Wissenschaft zu Leipzig, Philhist Classe, 1851, pp 351-352
724. Cfr. il Cap. IX. del pres. scritto.
725. Cod. th. 13, 3, 18 (= C. I. 12, 40, 8).
726. C , Institutions, II, 777, n. 2.
727. Cfr. S , o. c. I, 20 sgg. G , o. c. , 1 sgg. C , o. c. II, 777 sgg.
728. Su questi due codici, cfr. K , o. c. I, 941 sgg. K , Hist. des sources du droit romain, trad. fr., Paris, 1894, pp. 381 sgg. C , Storia delle fonti del diritto romano, Torino, 1909, pp. 114-116 e la bibliografia ivi citata.
729. A . M . 30, 4, 3 sgg.; 11: iuris professi scientiam repugnantium sibi legum abolevere discidia.
730. P . . 11, 20.
731. A . M . 30, 40, 8 sgg.
732. (Digest.) Const. Omnem, 1 sgg.
733. Nov. Theod. 1, 1 sgg.
734. Su l’opera giuridica di Teodosio II., cfr. K , o. c. I, 943 sgg.
735. Cod. th. 1, 1, 5.
736. Cod. th. 1, 1, 6.
737. Nov. Theod. 1; cfr. K , o. c. 1, 943 sgg.
738 Si tratta di una curiosa tradizione, che vale proprio la pena di riferire
P A , un letterato della prima metà del sec. XVI., fa, in un suo scritto (De exilio, Lipsiae, 1707, pp. 213-214), raccontare dal cardinal Giovanni de’ Medici, che, nella di lui biblioteca, era un libro di autore greco de rebus a Gothis in Italia gestis, in cui si diceva che Attila, allorquando ebbe invaso l’Italia, ordinò che niuno adoperasse più il latino e chiamò anzi dal suo paese maestri perchè insegnassero il gotico agli Italiani. Il T (o. c. II, 587-588) obbietta che Attila non poteva considerare l’Italia come cosa sua, e, quindi, legiferare secondo l’A riferirebbe. In verità, l’obbiezione non è insuperabile. Piuttosto, si potrebbe notare la stranezza del fatto che Attila avrebbe
imposto il gotico, anzichè l’unno, come lingua ufficiale. Ma ne anche a questa seconda obbiezione è impossibile replicare.
739. Const. Omnem 7 Cod. iust. 1, 17, 1, 10.
740. Ha dato di ciò una magistrale dimostrazione il Krumbacher, Gesch. d. byzant. Litteratur, München, 1897, 2ª ed., Einl. 1 sgg. Sui problemi di classificazione cronologica dell’antichità e del Medio Evo, discussi in questo breve paragrafo, cfr. G , Die Grenze zwischen Altertum u Mittelalter, in Kleine Schriften, Leipzig, 1894, V , 393 sgg
741 U , Anecdota Holderi, Bonn, 1877, p 67 M , Prooemium alle Variae di C , p VIII
742. U , o. c. 68 sgg. M , o. c. IX sgg.
743. Var. 9, 24, 8.
744. Var. 2, 3, 1 sgg.; 15, 4. 3, 33, 1 sgg. 10, 7, 2 sgg. etc.
745. La fonte è P (De bello goth. 1, 2), il quale però non riferisce la cosa come un fatto, della cui constatazione egli assuma la responsabilità, ma come un argomento dei nazionalisti Goti contro la figlia di Teodorico, Amalasunta
746. Cfr. C . Var. 1, 24.
747. C . Var. 10, 4, 6.
748. P . de bello goth. 1, 3.
749. C . Variae 1, 39, 4, 6 e, fors’anche, 2, 22. Cfr. M , Gesch. d. öst-gothischen Reiches in Italien, Breslau, 1824, p. 132, n. v.
750. C . Var. 5, 22. 4, 6.
751. in Nov. App. 7, 22.
752. C . Var. 2, 35.
753. I , Var. 1, 25.
754. C . Chron. ad a. 500.
755. I , Var. 1, 25; 28. 2, 7; 34; 39. 3, 29, 31. 4, 51. 7, 15.
756. C . Var. 7, 13.
757. E . Paneg. Theod. 56 e CDXXXVIII; cfr. M , o. c. 124 sgg.; 136 sgg.
758. C . Var. 8, 29; 30, 10, 30.
759. C . Var. 9, 24, 11.
760. C . Var. 8, 12, 8.
761. C . Var. 8, 18, 4.
762. P . de b. g. 1, 2; cfr. H , Italy and her invaders, Oxford, 1885, III, 585 sgg.
763. Debbono essere gli impiegati dell’officium a rationibus, cui spettava la cura suprema del fiscus; cfr. R , Fiscus in D R , Diz. ep. III, 133 sgg. H , Untersuchungen etc., pp. 29 sgg.
764. C . Var. 9, 21.
765. E . CDLII, 18, sgg. Fra i personaggi più colti dell’aristocrazia romana del tempo erano anche delle donne.
766. E . Paneg. Theod. 2; 76 C . De inst. dir. praef.
767. T , o. c. III, 51 sgg.
768. I , o. c. III, 35 sgg. M , Les moines d’Occident, Paris, 1860, II, 79-81.
769. A . (5, 14) si esprime testualmente: «Di quanti regnarono in Costantinopoli egli fu il primo sovrano assoluto, così di fatto, come di nome».
770. Sulla politica religiosa di Giustiniano, cfr. L , o. c. 144 sgg. S , o. c. I, 437 sgg. D , Justinien et la civilisation byzantine au VI. siècle, Paris, 1901, 552 sgg.
771. Cod. iust. 1, 5, 18, 4.
772. Cod. iust., 1, 11, 10, 2-3.
773. Z . 5, 5.
774. M . 18, O 187 d-e.
775. Su questo particolare, cfr. G , Gesch. d. Stadt d. Athen, I, 55-56 e 56, n. 1. Non mi è stato possibile avere tra mano il P (Ἱστορία τοῦ Ἔλλ. ἔθνους, 1887), ove, secondo trovo indicato, si nega la realtà delle soppressioni avvenute nel 529, tesi questa, che però non è stata accolta dai più recenti storiografi di quell’età
776 A 2, 30 M 18, O 237-238
777 A 2, 28; 30-31 Sulla fine della Università ateniese, cfr anche Z , o c 59 sgg
778 Z , o c 63 Z , o c III4 , 2, 917, n 1
779 M 18, O 187 d-e Il cronista fa tale divieto contemporaneo all’altro dell’insegnamento della filosofia Ma questo è impossibile Nel 529 Giustiniano aveva già riconosciuto quelle scuole (M 18, O 183) L’ordine della chiusura della facoltà di giurisprudenza deve essere quindi contemporaneo alla pubblicazione del Digesto (Const. Omnem 7).
780 I Or 3, p 153
781 C , Anecd graeca e Codd Paris IV, 315
782 P H A 26 (= P 74 c-d)
783 Su Procopio, quale fonte della storia di Giustiniano, cfr H , Zur Beurtheilung d Geschichtsschreibers Procopius, Munich, 1896 B , Zur Beurtheil Procopius, Ansbach, 1896, C , o c V, 1018-1019 K , o. c. I2 , 230-237 B , o. c. I, 359 D , o. c. XII sgg.
784. K , o. c. I2 , 373.
785. Z . 14, 6, 31-32 (= P. 2. II. 63, b.)
786. in Nov. App. 7, 22.
787. Cod. iust. 2, 7, 22, 4-5; 24, 4-5.
788. Cod. iust. 2, 7, 11, 1 sgg.
789. Sull’opera giuridica di Giustiniano, cfr. G , o. c. VIII, 30 sgg. K , o. c. I, 1003 sgg. K , o. c. p. 431 sgg. D , o. c. 250 sgg C , o c 130 sgg , ove, assai più del testo è pregevole il copioso apparato bibliografico
790 Const Deo auctore 5
791. Const. Tanta, 11.
792. Const. Tanta 13.
793. Const. Imper. maiest., praef.; cfr. De Iust. cod. conf. praef.
794. Const. Imper. maiest. 3.
795. Ibid. 7.
796. Const. Imper. maiest. 3.
797. Const. Omnem 7. Giustiniano (ibid.) soggiunge che tale investitura ufficiale fu, dai suoi predecessori, data anche a Berito, a Roma e a Costantinopoli, ma non ad altri luoghi. Egli dimentica però le costituzioni imperiali, cui si riferisce un passo del Digesto (27, 1, 6, 12), secondo cui i principi riconoscono l’insegnamento della giurisprudenza nelle province, pur non onorandone i maestri delle consuete immunità: «qui ius civile docent in provincia vacationem non habent, Romae docentes habent »
798. Cotali sedi di scuole giuridiche non dovevano essere poche; cfr. Dig. 27, 1, 6, 12 e B , Rechtslehrer u Rechtsschulen, 71 sgg
799. Const. Omnem 7.
800. Cfr. il § IX. del pres. capitolo.
801. Cfr. S , o. c. I, 263 n. a. K , o. c. I, 1023. A Berito dovevano esservene certamente più di due. Durante i lunghi anni di compilazione delle Pandette, noi troviamo nella Commissione due professori di Berito, i quali, naturalmente, erano costretti a soggiornare a Costantinopoli Se a Berito non ve ne fossero stati altri, quella gloriosa facoltà giuridica sarebbe rimasta senza maestri
802 Per la compilazione della prima edizione del Codice v’è solo un professore di Costantinopoli; per la seconda, solo uno di Berito; per le
Pandette, due di Costantinopoli e due di Berito; per le Istituzioni, uno di Costantinopoli e uno di Berito.
803. Significava Dupondii studenti da due dramme? E in che modo a codesto nome si convenivano le critiche imperiali? Cfr., su codesta oscura, questione, P , Miscellanea, I, 107 sgg R , in Zeitschrift f Rechtsgeschichte, III, 38
804 Const Omnem 1
805. Const. Omnem 7.
806. Const. Omnem 5.
807. Const. Omnem 9-10.
808. Const. Omnem 10. Non è inopportuno rilevare l’analogia di queste disposizioni con quelle che regolano le Università medievali, di cui fu modello Bologna (S , o. c. I, 556-557).
809. Il testo, come in altri punti della costituzione, ha leges, ma sul significato della parola, cfr. K , o. c. 468, n. 1.
810. Giustiniano dice partes legum, ma cfr. K , l. c. K , o. c. I, 1026.
811. Const. Omnem, 1; 4.
812. Ibid. 1; 5.
813. Const. Omnem 1.
814. Sul probabile valore simbolico di questa, come di parecchie altre cifre, contenute in questi programmi, cfr. B , o. c. I, 368-369.
815. Const. Omnem 2 sgg.
816. I . L , De magistr. 3, 29.
817. H , Commentarius de Ioanne Laurentio Lydo, p. IX, nell’edizione Bonnense delle opere di Lido.
818. l. c. L’H rimane incerto fra la lingua greca e la latina, ma il testo del decreto fa propendere per quest’ultima: Giustiniano lo lodava per la sua perizia nella ρωμαίων φωονὴ (L . De magistr. 3, 29).
819. Cfr. A . V . Caes. 10, 1.
820. C . De rep. 4, 3, 3.
821. Cfr. B , Scuola, Stato e politica in Roma repubblicana, in Riv. di filol. class., 1910, fasc. 4º.
822. Δημηγωρία etc. (in T . Orationes, ed. D ) p. 21 b-c.
823. S . Ep. 1, 79.
824. Const. Omnem, 7.
825. Const. Omnem 10.
826. Cfr. H , o. c. 227 e sgg. G , Kulturgesch. d. Kaiserzeit, Stuttgart, 1903, I, 141.
827. Cfr. P . o. c. 84.
828. C. I. 10, 53, 2.
829. È tipica la legge del Cod. th. 13, 3, 1.
830 Cfr Cod th 12, 2, 1 (= C I 10, 37 (36))
831 Const Omnem 7
832 CIL 8, 20 684
833 Cfr C , Procurator in D S , Dict d’ant class 4, 1, p 662
834 CIL 10, 1739
835 Cfr in ispecie il CIL 10, 7580; 14, 2916
836 CIL 6, 2132
837 Così hanno opinato, contro il M (in H G , Texte u Unters etc 1903, 111-112), l’H , o c 305 nota e il L , o c 140-141 Ma, fuori di Roma, gli imperatori avevano certamente biblioteche private ne è prova quella greco-latina, collocata da Adriano nella sua villa a Tivoli , e, se questo accadeva fuori di Roma, doveva a potiori avvenire in Roma e in Costantinopoli
