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Methods in Molecular Biology 2242

Bacterial Pangenomics

Methods

and Protocols

Second Edition

M ETHODSIN M OLECULAR B IOLOGY

SchoolofLifeandMedicalSciences

UniversityofHertfordshire Hatfield,Hertfordshire,UK

Forfurthervolumes: http://www.springer.com/series/7651

Forover35years,biologicalscientistshavecometorelyontheresearchprotocolsand methodologiesinthecriticallyacclaimed MethodsinMolecularBiology series.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-bystepfashion,openingwithanintroductoryoverview,alistofthematerialsandreagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported withahelpfulnotessectionofferingtipsandtricksofthetradeaswellastroubleshooting advice.ThesehallmarkfeatureswereintroducedbyserieseditorDr.JohnWalkerand constitutethekeyingredientineachandeveryvolumeofthe MethodsinMolecularBiology series.Testedandtrusted,comprehensiveandreliable,allprotocolsfromtheseriesare indexedinPubMed.

BacterialPangenomics

MethodsandProtocols

SecondEdition

DepartmentofBiology,UniversityofFlorence,SestoFiorentino,Firenze,Italy

Editors

AlessioMengoni

DepartmentofBiology

UniversityofFlorence SestoFiorentino,Firenze,Italy

MarcoFondi

DepartmentofBiology

UniversityofFlorence SestoFiorentino,Firenze,Italy

GiovanniBacci

DepartmentofBiology

UniversityofFlorence SestoFiorentino,Firenze,Italy

ISSN1064-3745ISSN1940-6029(electronic)

MethodsinMolecularBiology

ISBN978-1-0716-1098-5ISBN978-1-0716-1099-2(eBook) https://doi.org/10.1007/978-1-0716-1099-2

© SpringerScience+BusinessMedia,LLC,partofSpringerNature2021

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Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse.

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Preface

Bacterialgenomicshasbecomeinthelast10yearsamatureresearchfield,contributedby ecologists,geneticists,bacteriologists,molecularbiologists,andevolutionarybiologists.In 1999,CarlWoesewrote“Genomesequencinghascomeofage,andgenomicswillbecome centraltomicrobiology’sfuture.Itmayappearatthemomentthatthehumangenomeisthe mainfocusandprimarygoalofgenomesequencing,butdonotbedeceived.Therealjustificationinthelongrun,ismicrobialgenomics”[1].Indeed,microbialgenomics,especially prokaryoticgenomics,isnowcentralinlifescience,spanningfromenvironmentalstudies tomedical,agricultural,andindustrialapplication.Thediscoveryoftheimportanceof microbialcommunities,theirtremendousdiversity,andtheirimpactoverthelifeofmulticellularorganisms,includinghumans,hasledtotheemergenceofnewconcepts,including evolutionaryinterpretationsofbioticrelationships.Concurrentimprovementofsequencing technologiesisthenallowingtoshifttheinterestfromthemereassembly/reconstruction anddescriptionofgenomesandmetagenomestotheirfunctionalinterpretation.

Underthesepremises,thissecondeditionofthebook BacterialPangenomics belonging tothe MethodsinMolecularBiology series,becameachallenge,intheaimtoproposeto readersselectedup-to-datemethodswhicharerelevantandcanbringforthnoveldiscoveries insucharapidlyevolvingfield.Consequently,thebookhasbeencompletelyrenewedwith respecttothepreviousedition,payingspecialattentiontothetechnicalandcomputational improvements,tothemethodsusedforbacterialpangenomeanalysiswhichrelieson microbiomestudiesandmetagenomicdata,andalsoconsideringthenecessityofcomputationalmethodsbeingunderstandabletoeveryresearcherinthefield,notadomainrestricted tobioinformaticians.

Thisbookhasbeenorganizedintofivemainparts,startingfromtheup-to-date sequencingmethods(PartI)tothemethodsfordeepphylogeneticanalysis(PartII),to thecentralroleofmetagenomicdatainunderstandingthegenomicsofthemanyyet unculturedbacteria(PartIII),tothecurrentPartIV.Thisbookendswithtwochapters devotedtopromotingthediffusionofcomputationalgenomictoolsamonggraduateand undergraduatestudents(PartV).

Theaimofthepresentbookisthen,asforthepreviousedition,toserveasa“field guide”bothforqualifiedinvestigatorsonbacterialgenomicsandforlessexperienced researchers(includingstudentsandteachers)whoneedreferencesforapproachinggenomic analysisandgenomedata.

Preface.. ...................................................................v

Contributors .................................................................ix

PART IOPPORTUNITIESFROM NOVEL SEQUENCING TECHNOLOGIES

1PacBio-BasedProtocolforBacterialGenomeAssembly ......................3 AgataMotyka-Pomagruk,SabinaZoledowska,MichalKabza, andEwaLojkowska

2TheIlluminaSequencingProtocolandtheNovaSeq6000System ............15 AlessandraModi,StefaniaVai,DavidCaramelli,andMartinaLari

PART IIPANGENOMICSOF CULTURED ISOLATES

3ComparativeAnalysisofCoreandAccessoryGenesinCoexpression Network... ............................................................45 BiliangZhang,JianJiao,PanZhang,Wen-JingCui, ZidingZhang,andChang-FuTian

4InferringCoreGenomePhylogeniesforBacteria ...........................59 AlexanderKellerandMarkusJ.Ankenbrand

5InferringPhylogenomicRelationshipofMicrobesUsingScalable Alignment-FreeMethods... .............................................69 GuillaumeBernard,TimothyG.Stephens,Rau ´ lA.Gonza ´ lez-Pech, andCheongXinChan

6FastPhylogenyReconstructionfromGenomesofClosely RelatedMicrobes... ....................................................77 BernhardHauboldandFabianKlotzl

7ComparativeGenomics,fromtheAnnotatedGenometoValuable BiologicalInformation:ACaseStudy .....................................91 SabinaZoledowska,AgataMotyka-Pomagruk,AgnieszkaMisztak, andEwaLojkowska

PART IIIDARK MATTER PANGENOMICS

8AccurateAnnotationofMicrobialMetagenomicGenesandIdentification ofCoreSets ............................................................115 ChiaraVanni

9MetagenomicAssembly:ReconstructingGenomesfromMetagenomes ........139 ZhangWang,Jie-LiangLiang,Li-NanHuang, AlessioMengoni,andWen-ShengShu

10GenomeRecovery,FunctionalProfiling,andTaxonomicClassification fromMetagenomes. ....................................................153 DavideAlbaneseandClaudioDonati

viiiContents

11FunctionalMetagenomicsforIdentificationofAntibiotic ResistanceGenes(ARGs)... .............................................173 FrancescaDiCesare

12HostTraitPredictionfromHigh-ResolutionMicrobialFeatures ..............185 GiovanniBacci

PART IVPROGRESSESIN GENOME-TO-PHENOME INFERENCE

13PhylogeneticMethodsforGenome-WideAssociationStudiesinBacteria ......205 XavierDidelot

14Simple,Reliable,andTime-EfficientManualAnnotation ofBacterialGenomeswithMAISEN. .....................................221 MikolajDziurzynski,PrzemyslawDecewicz,KarolCiuchcinski, AdrianGorecki,andLukaszDziewit

PART VCOOKBOOKFOR PANGENOMICS

15ACompendiumofBioinformaticToolsforBacterialPangenomics toBeUsedbyWet-LabScientists .........................................233 CamillaFagorziandAliceCheccucci

16AProtocolforTeachingBasicNextGenerationSequencing(NGS) AnalysisSkillstoUndergraduateStudentsUsingBashandR.. ...............245 MarcoFondiandGiovanniBacci Index... ...................................................................267

Contributors

DAVIDE ALBANESE • UnitofComputationalBiology,ResearchandInnovationCentre, FondazioneEdmundMach,SanMicheleall’Adige,Italy

MARKUS J.ANKENBRAND • CenterforComputationalandTheoreticalBiology,Biocenter, UniversityofWu¨rzburg,Wu¨rzburg,Germany;DepartmentofBioinformatics,Biocenter, UniversityofWu¨rzburg,Wu¨rzburg,Germany;ComprehensiveHeartFailureCenter, UniversityHospitalWu¨rzburg,Wu¨rzburg,Germany

GIOVANNI BACCI • DepartmentofBiology,UniversityofFlorence,SestoFiorentino,Italy

GUILLAUME BERNARD • SorbonneUniversite´s,UPMCUniversite´Paris06,Institutde BiologieParis-Seine(IBPS),Paris,France

DAVID CARAMELLI • DepartmentofBiology,UniversityofFirenze,Firenze,Italy

CHEONG XIN CHAN • InstituteforMolecularBioscience,TheUniversityofQueensland, Brisbane,QLD,Australia;SchoolofChemistryandMolecularBiosciences,TheUniversityof Queensland,Brisbane,QLD,Australia;AustralianCentreforEcogenomics,The UniversityofQueensland,Brisbane,QLD,Australia

ALICE CHECCUCCI • DepartmentofAgriculturalandFoodScience,UniversityofBologna, Bologna,Italy

KAROL CIUCHCINSKI • FacultyofBiology,InstituteofMicrobiology,Departmentof EnvironmentalMicrobiologyandBiotechnology,UniversityofWarsaw,Warsaw,Poland

WEN-JING CUI • StateKeyLaboratoryofAgrobiotechnology,andCollegeofBiological Sciences,ChinaAgriculturalUniversity,Beijing,China

PRZEMYSLAW DECEWICZ • FacultyofBiology,InstituteofMicrobiology,Departmentof EnvironmentalMicrobiologyandBiotechnology,UniversityofWarsaw,Warsaw,Poland

FRANCESCA DI CESARE • MagneticResonanceCenter(CERM),UniversityofFlorence,Sesto Fiorentino,Italy;DepartmentofBiology,UniversityofFlorence,Florence,Italy

XAVIER DIDELOT • SchoolofLifeSciencesandDepartmentofStatistics,Universityof Warwick,Coventry,UK

CLAUDIO DONATI • UnitofComputationalBiology,ResearchandInnovationCentre, FondazioneEdmundMach,SanMicheleall’Adige,Italy

LUKASZ DZIEWIT • FacultyofBiology,InstituteofMicrobiology,Departmentof EnvironmentalMicrobiologyandBiotechnology,UniversityofWarsaw,Warsaw,Poland

MIKOLAJ DZIURZYNSKI • FacultyofBiology,InstituteofMicrobiology,Departmentof EnvironmentalMicrobiologyandBiotechnology,UniversityofWarsaw,Warsaw,Poland

CAMILLA FAGORZI • DepartmentofBiology,UniversityofFlorence,Florence,Italy

MARCO FONDI • DepartmentofBiology,UniversityofFlorence,SestoFiorentino,Italy

RAU ´ L A.GONZA ´ LEZ-PECH • InstituteforMolecularBioscience,TheUniversityofQueensland, Brisbane,QLD,Australia

ADRIAN GORECKI • FacultyofBiology,InstituteofMicrobiology,Departmentof EnvironmentalMicrobiologyandBiotechnology,UniversityofWarsaw,Warsaw,Poland

BERNHARD HAUBOLD • ResearchGroupBioinformatics,Max–Planck-Insitutfu¨r Evolutionsbiologie,Plo¨n,Germany

LI-NAN HUANG • SchoolofLifeSciences,SunYat-SenUniversity,Guangzhou,Guangdong Province,China

JIAN JIAO • StateKeyLaboratoryofAgrobiotechnology,andCollegeofBiologicalSciences, ChinaAgriculturalUniversity,Beijing,China

MICHAL KABZA • DepartmentofIntegrativeGenomics,FacultyofBiology,AdamMickiewicz University,Poznan,Poland

ALEXANDER KELLER • CenterforComputationalandTheoreticalBiology,Biocenter, UniversityofWu¨rzburg,Wu¨rzburg,Germany;DepartmentofBioinformatics,Biocenter, UniversityofWu¨rzburg,Wu¨rzburg,Germany

FABIAN KLOTZL • ResearchGroupBioinformatics,Max–Planck-Insitutfu¨r Evolutionsbiologie,Plo¨n,Germany

MARTINA LARI • DepartmentofBiology,UniversityofFirenze,Firenze,Italy

JIE-LIANG LIANG • InstituteofEcologicalScience,SchoolofLifeScience,SouthChinaNormal University,Guangzhou,GuangdongProvince,China

EWA LOJKOWSKA • DepartmentofPlantProtectionandBiotechnology,IntercollegiateFaculty ofBiotechnologyUniversityofGdansk&MedicalUniversityofGdansk,Universityof Gdansk,Gdansk,Poland

ALESSIO MENGONI • DepartmentofBiology,UniversityofFlorence,Florence,Italy

AGNIESZKA MISZTAK • DepartmentofPlantProtectionandBiotechnology,Intercollegiate FacultyofBiotechnologyUniversityofGdansk&MedicalUniversityofGdansk,University ofGdansk,Gdansk,Poland

ALESSANDRA MODI • DepartmentofBiology,UniversityofFirenze,Firenze,Italy

AGATA MOTYKA-POMAGRUK • DepartmentofPlantProtectionandBiotechnology, IntercollegiateFacultyofBiotechnologyUniversityofGdansk&MedicalUniversityof Gdansk,UniversityofGdansk,Gdansk,Poland

WEN-SHENG SHU • InstituteofEcologicalScience,SchoolofLifeScience,SouthChinaNormal University,Guangzhou,GuangdongProvince,China;GuangdongMagigene BiotechnologyCo.Ltd.,Guangzhou,GuangdongProvince,China

TIMOTHY G.STEPHENS • InstituteforMolecularBioscience,TheUniversityofQueensland, Brisbane,QLD,Australia

CHANG-FU TIAN • StateKeyLaboratoryofAgrobiotechnology,andCollegeofBiological Sciences,ChinaAgriculturalUniversity,Beijing,China

STEFANIA VAI • DepartmentofBiology,UniversityofFirenze,Firenze,Italy

CHIARA VANNI • MaxPlanckInstituteforMarineMicrobiology,Bremen,Germany;Jacobs University,Bremen,Germany

ZHANG WANG • InstituteofEcologicalScience,SchoolofLifeScience,SouthChinaNormal University,Guangzhou,GuangdongProvince,China

BILIANG ZHANG • StateKeyLaboratoryofAgrobiotechnology,andCollegeofBiological Sciences,ChinaAgriculturalUniversity,Beijing,China

PAN ZHANG • StateKeyLaboratoryofAgrobiotechnology,andCollegeofBiologicalSciences, ChinaAgriculturalUniversity,Beijing,China

ZIDING ZHANG • StateKeyLaboratoryofAgrobiotechnology,andCollegeofBiological Sciences,ChinaAgriculturalUniversity,Beijing,China

SABINA ZOLEDOWSKA • DepartmentofPlantProtectionandBiotechnology,Intercollegiate FacultyofBiotechnologyUniversityofGdansk&MedicalUniversityofGdansk,University ofGdansk,Gdansk,Poland;InstituteofBiotechnologyandMolecularMedicine,Gdansk, Poland

OpportunitiesfromNovelSequencingTechnologies

PacBio-BasedProtocolforBacterialGenomeAssembly

AgataMotyka-Pomagruk,SabinaZoledowska,MichalKabza, andEwaLojkowska

Abstract

Acquisitionofhigh-qualitybacterialgenomesisfundamental,whilehavinginmindinvestigationofsubtitle intraspeciesvariationinadditiontodevelopmentofsensitivespecies-specifictoolsfordetectionand identificationofthepathogens.Inthisview,PacificBiosciencestechnologyseemshighlytemptingtaking intoconsiderationover10,000bplengthofthegeneratedreads.Inthiswork,wedescribeabacterial genomeassemblypipelinebasedonopen-sourcesoftwarethatmightbehandledalsoby non-bioinformaticiansinterestedintransformationofsequencingdataintoreliablebiologicalinformation. Withtheuseofthismethod,wesuccessfullyclosedsix Dickeyasolani genomes,whiletheassemblyprocess wasrunjustonaslightlyimproveddesktopcomputer.

Keywords Nextgenerationsequencing,Whole-genomesequencing,Single-moleculereal-time sequencing,Softrotbacteria, Dickeya spp., Pectobacterium spp.

1Introduction

Highthroughputandlowcostarethehallmarksofnextgeneration sequencing(NGS)methods,whichreplacedSanger-based approachesinnumerousstudiesonwholebacterialgenomes.In early2011,PacificBiosciences(PacBio)RSsequencerhasbeen releasedtogetherwithIonTorrent’sPGMandtheIllumina MiSeqplatforms[1].ThefirsttechniqueexploitsDNApolymerase asasingle-moleculereal-time(SMRT)sequencingengine,yielding significantlylongerreads(approx.10,000bp)thantheother above-mentionedmethods.Besides,trackingwiththebase-pair resolutionthekineticparametersofeachindividualenzymeopens perspectiveforstudyingthebasemethylationpatterns,polymerase inhibitors,orDNAbindingproteins[2].

Theinsightintosinglenucleotideincorporationsaccordingto SMRTtechnologyisachievedwithinzero-modewaveguide (ZMW),beingananophotonicvisualizationcompartment operatingat100zeptolitersdetectionvolumes[3].Asdepictedin

AlessioMengonietal.(eds.), BacterialPangenomics:MethodsandProtocols,MethodsinMolecularBiology,vol.2242, https://doi.org/10.1007/978-1-0716-1099-2_1, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2021

Fig.1 TheprincipleofPacBiosequencingtechnology.PolymeraseimmobilizedatthebottomofZMVis incorporatingfluorescentphospholinkednucleotidesintothenascentDNAstrand.Attachmentofeachcorrect nucleotideisassociatedwithspecificlightemissionsignalfollowedbyliberationofthefluorophore. Subsequently,theenzymeshiftstothenextnucleotideuntiltheendofthetemplatestrandisreached. Brownarrowsmarkexcitationlight,whileblueandyellowarrowsrefertotheemissionspectra,corresponding inthiscasetoTandG,respectively

Fig. 1,DNApolymeraseattachedtoZMWbuildsacomplementary strandtotheDNAmatrixfromfluorescentphospholinkednucleotidesinthepresenceofaprimer.Duetodiverseexposuretimeto ZMW,approachingoffluorescentlylabelednucleotidesiseasily discriminatedfromactualprocessingofthematchingtagged dNTPs.Notablepeakinthefluorescentsignalisnotedwhenthe α-β phosphodiesterbondiscleavedwiththereleaseofafluorophore.Usageoffourdistinctfluorescentlabelspermitstodecipher whichnucleotide,namely,dATP,dTTP,dGTP,ordCTP,hasbeen introducedtoastatedpositioninanewlysynthesizedstrand.The polymerasecontinueselongationofthenascentDNAafterhaving shiftedbyonebase[2, 3].

Apartfromthealreadymentioneddroppingpricesalsohigher availabilityofNGSplatformscontributedtothefactthatthenumberofstudiestargetingacquisitionofwholegenomicinformation isconstantlyincreasing.Forinstance,therewere5,800sequencing projectsregisteredinTheGenomesOnlineDatabase(GOLD)in 2009[4],incontrasttothecurrentpopularityofsuchactions reachingthenumberof317,275(October,2019).Still,vastmajorityofthegenomesdepositedinpubliclyavailabledatabasesarein lowqualitydraftstatesasthefinishingstepsareconsideredproblematic,time-consumingandlaborious[5].

Though,iftheresearchisaimedatsequencingandsubsequent comparisonofthegenomesofmultiplestrainsclassifiedwithina

certainspecies,high-qualitygenomicassembliesarerequiredto revealsubtleintraspeciesvariation[6].

Scientificinterestofourgroupisfocusedonphytopathogens, especiallypectinolyticplantpathogenicbacteriaclassifiedtothe genera Dickeya and Pectobacterium withintherecentlyestablished family Pectobacteriaceae [7].Thesemicroorganismscausesoftrot ondiversecrops(especiallypotatotubers),vegetablesandornamentalsinadditiontoblacklegsymptomsthatarerestrictedto potatoplants[8].Significanteconomiclosses,forexample,reaching30millioneuroeachyearjustintheseedpotatoproduction sector[9],resulteitherfromtubertissuedecayorrejection/downgradingofthelatentlyinfectedplantingmaterial.Duringourpreviousstudies,wecollectedapoolofpectinolyticbacteriabelonging tothe Dickeyasolani and Pectobacteriumparmentieri speciesthat differednotablyintheirvirulence;bothinthecapacitytomacerate planttissueandtheabilitytoproducevirulencefactors[10–12].Therefore,wedecidedtoincorporatecomparativegenomic approachestoscreentheuniqueandaccessorypangenomefractionsoftworecentlyestablishedpectinolyticspecies, D.solani and P.parmentieri,forthepresenceofgenesdiscriminatinghighlyfrom lowvirulentstrains[13, 14].Regarding D.solani,atremendous efforthasbeenmadeinordertoproposeagenomeassembly pipelineyieldingsequencesofsufficientqualityfortheintended downstreamapplications.Previousattempttakingadvantageof both454pyrosequencing(191,539reads)andPacBioSMRT (118,344reads)technologiesledtoaquisitionofadraftgenomic sequenceof D.solani IFB0099strainthatconsistedof97contigs [15].Inthatstudy,454readswereconvertedtoFastQCformat andsubsequentlytrimmedwiththeuseofStreamingTrimversion 1.0[16].Hybridassemblyofthetrimmed454readswithPacBio datawasachievedwithpacBioToCAandtherunCAprogramofthe Celeraassembler[17].Prokkaversion1.7.2[18]wasappliedfor functionalannotation[15].Though,theresultantlowaccuracy draftstatusofthegenometurnedouttobeinsufficientforthe needsoftheplannedcomparativegenomicstudy.Therefore,we developedanovelgenomeassemblypipelineproveneffectiveon ten D.solani strains(Table 1).ItrequiredobtainingsolelyPacBio reads(Table 2),tookadvantageofopen-sourcesoftwareandrunon justslightlyimproveddesktopcomputerhandledbynonbioinformaticians.Inthiswayweclosedsix D.solani genomes,whilethe othersremaininjustfewscaffolds(Table 3).As D.solani isahighly homogeneousspecies,itmightbealsointerestingforother researcherstolookupthealternativehybridapproach(basedon PacBioandIlluminareads)thatweincorporatedforassemblingthe genomesof P.parmentieri [13],beingamuchmoreheterogeneous species.

Table1 Dickeyasolani strainsforwhichthehereindescribedPacBioreads-basedgenomeassemblypipeline wasused

Strainnos.Countryofisolation,yearOriginReferences

IFB0099a (IPO2276,LMG28824) Poland,2005Potato,cv.Santa[19, 20]

IFB0167Poland,2009Potato,cv.Fresco[11]

IFB0212Poland,2010Potato[21]

IFB0223a (457) Germany,2005Rhizosphereofpotato[10]

IFB0231 (VIC-BL-25) Finland,NAPotato,cv.Victoria[22]

IFB0311Poland,2011Potato,cv.Innovator[11]

IFB0417Portugal,2012Potato,cv.LadyRosetta[23]

IFB0421Portugal,2012Potato,cv.LadyRosetta[23]

IFB0487Poland,2013Potato,cv.Vineta[23]

IFB0695Poland,2014Potato,cv.Arielle[23]

IFBBacterialstrainscollectionofIntercollegiateFacultyofBiotechnologyUniversityofGdanskandMedicalUniversity ofGdansk,Gdansk,Poland Numbersattributedtotheanalyzed D.solani strainsinotherbacterialcollectionsaredepictedinparentheses aThegenomesofthesetwostrainshavebeensequencedintheframesoftheformerprojectbyBaseClear(Leiden,The Netherlands)company(see Note2)

2Materials

2.1GeneralPurpose NanoDrop1000Spectrophotometer(ThermoScientific,USA).

2.2Biological Material

2.3Computer Hardware

2.4Computer Software

3Methods

3.1Obtaining ofPacBioReads

Bacterialbiomass(thestrainsanalyzedhereinarelistedinTable 1) or8 μgofhighmolecularRNA-freeDNA(see Note1).

SystemLinuxMintversion17.3Cinnamon64-bit,Memory62.8 GiB,HardDrives190GB,ProcessorIntelCorei7-5820K 3.30GHzx6,NVIDIACorporationGM206GeForceGTX960.

SMRTAnalysisversion2.3,Canuversion1.5[24],Quiver[25], Prokkaversion1.12[18],samtools,pbalign.

l Bacterialbiomassof8 D.solani strainsspreadinreductivemanneronLysogenyAgarmediumhasbeensenttoGATCBiotech (Konstanz,Germany)(see Note2)formultiplicationofthis

Table2

PacBioreadsgeneratedforthestudied Dickeyasolani genomes

Strain

IFB0167150,2921,407,716,05122,946936693,3631,348,072,19823,10314,439258,2621,340,261,63761685189

IFB0212150,292321,139,80524,438213626,205309,536,32424,76311,81262,031307,660,28560474959

IFB0231300,584805,230,98821,411267871,357773,018,93321,74110,833170,468766,788,39555004498

IFB0311300,584858,690,22224,096285673,736825,635,13724,43311,197221,577816,213,96043143683

IFB0417150,2921,107,402,39614,9837368102,7941,043,716,23815,12810,153149,1461,041,633,97094846983

IFB0421150,2921,234,534,05117,4678214106,3881,201,169,37517,65011,290186,0601,197,494,23989136436

IFB0487150,2921,080,552,37714,4137189100,2741,029,957,36614,60310,271145,8661,027,880,86895247046

IFB0695150,2921,159,232,65816,064771399,6041,127,327,57516,29411,318160,2711,124,511,59495057016

a CharacteristicsofthereadsgeneratedforIFB0099andIFB0223strainsbyBaseClear(Leiden,TheNetherlands)companyarelistedin Note2

b 300,584readsweregeneratedby2SMRTcells2movies,while150,292readswereobtainedfrom1SMRTcell1movie

N 50 -minimumlenghtofcontigscoveringhalfoftheassemblybasses.

Table3

Statisticsoftheassembled Dickeyasolani genomes

AssemblystatisticsAnnotationstatistics

No.of genes

Largestcontig (bp)N

Genomesize (bp)

No.ofN bases

No.of scaffolds

Genome

IFB0099104,932,9204,932,9204,932,920156.244326416422751

IFB0167104,922,2894,922,2894,922,289156.254308414622751

IFB0212204,909,9353,946,0103,946,010156.254304414318721

IFB0223104,937,5544,937,5544,937,554156.244328416722751

IFB0231104,924,7024,924,7024,924,702156.244313415122751

IFB0311304,913,2612,394,2831,850,246256.244306414420741

IFB0417104,924,1024,924,1024,924,102156.244608444622751

IFB0421104,934,5374,934,5374,934,537156.244349418722751

IFB0487404,882,1243,440,8323,440,832156.234572440922751

IFB0695704,904,7692,442,930755,734256.254337417222751

N 50 -minimumlenghtofcontigscoveringhalfoftheassemblybasses.L 50 -numberofcontigscontaininghalfoftehassembledgenome.

Fig.2 PipelineforgenerationofPacBioreads.DNAisfragmented.Then fragmentsoftheselectedsizearerepairedandligatedtoSMRTbelladapters. TheprimersandsubsequentlyDNApolymeraseannealtoSMRTbelltemplates. AfterbindingtoZMVs,theSMRTbelltemplatesaresequencedonPacBioRSII platform.Thegeneratedreadsaredeprivedofadapters,shortandlowquality sequencestoformasetoffilteredsubreads

materialinLysogenyBrothmediumat28 C,celldisruption/ lysis,isolationofhighmolecularweightDNA(see Note1)with MaxiPrepQiagen(Hilden,Germany),performingquantificationandqualitycontroloftheisolatedgenomicDNA.

l SubsequentlystandardgenomicPacBiolibrarywasprepared (Fig. 2, see Note3)involving:fragmentationofDNA (10,000bp, see Notes4 and 5),sizeselection,repairofDNA damage(see Note6)andDNAends(see Note7)andadapter ligation(see Note8),annealingofprimertoSMRTbelltemplates(see Note9),andannealingofpolymerasetoSMRTbell templates(see Note10)[26, 27].

l SMRTbelltemplatesareincorporatedintoZMWs(see Note11).

3.2Primary Analysis:Quality ControlandAdapter TrimmingwithSMRT Analysis

3.3Assembly ofPacBioData

3.4Polishing theAssembled Genomes withPacBioData

3.5Functional Annotation oftheGenomes

4Notes

l SequencingonPacBioRSIIplatformproceedswiththeuseof rawdatapackage(see Note12,Fig. 1).

l ThereadsgeneratedbythePacBioRSIIinstrumentwereprocessedandfilteredwiththeuseofSMRTAnalysisversion2.3 software(see Note13)togeneratethesubreads(Fig. 2; Table 2).DataManagementandSMRTAnalysismodulesare compatiblewiththePacBioRSIIdataofinterest.

l AtfirstalltheFASTQformatfileshavebeenmerged(see Note 14).

l ThenthesuccessorofCeleraassembler,Canuversion1.5[24]was incorporatedforfurthercorrection,trimmingandsubsequent assemblyofthePacBioreads.Thissoftwareisbasedonhierarchical strategy,thereforeprofitsfrommultipleroundsofreadoverlappinginordertoincreasethequalityofsingle-moleculereads beforeperformingtheassemblyprocess(see Note15).

l Primarilythepacbio.fofn(see Note16)filecontainingallpaths to.bas.h5(see Note17)filespresentinthepacbiofolderwas created.

l Secondly,thedatawereconvertedwithsamtools(version1.4.1) faidxandpbalign[28](see Notes18 and 19).

l ForgettingconsensusandvariantcallingQuiver[25]was applied(see Note20).

l FunctionalannotationwasaccomplishedwiththeuseofProkka version1.12[18](see Note21;Table 3).

1.DNAconcentrationshouldexceed80ng/μlwithamaximum volumeof100 μl.HighDNApurityisrequired,meaningOD 260/280 1.8andOD260/230 1.9.Itisimportantthat thesampledoesnotcontainimpurities,suchasbiological macromolecules(RNA,proteins,polysaccharides,lipids),chelatingagents(e.g.,EDTA),divalentmetalcations(Mg2+),any detergents(SDS,TritonX-100),ordenaturants(e.g.,phenol, guanidiniumsalts),andhasbeensuspendedinRNase-,DNase,andprotease-freeTris–HClbuffer(pH8.0–8.5).Usually,an additionalsample(e.g.,20 μl)oftheDNAelutionbufferused isrequestedbythesequencingcompanies.DNAshouldnot

havebeensubjectedtomultiplefreeze-thawingcycles,high temperatures(e.g., >65 Cforover1h),UVlight,orpH extremes(< 6or >9).DNAshouldratherhavebeenextracted withtheuseofcommercialkitsastheorganicextractionmethods(involvingphenolorTRIzol™)mayinhibittheenzymes utilizedduringlibrarypreparation.DNAmaybeshippedatthe roomtemperature,howeverperformingtransferoftherefrigeratedmaterialishighlyrecommended[26, 27].

2.Regardingtwo D.solani strains,IFB0099andIFB0223,high qualitygenomicdsDNAwassentintheframesoftheprevious projecttoanothersequencingcompanyBaseClear(Leiden, TheNetherlands).ForIFB0099andIFB0223,respectively, thefollowingreads’statisticshavebeenacheived:numberof reads118,344and102,248;sampleyield395,000,000bpand 356,000,000bpinadditiontoaveragelength3,340,000bp and3,490,000bp.

3.SMRTbellTemplatePrepKitisusedforlibrariespreparation, whileAMPure® PBbeadsareneededforthepurificationsteps. Thetemplatepreparationprotocollasts3–6h.

4.CharacteristicsoftheintroducedDNAsample,namely,organismoforigin,quality,amount,andpurity,arecrucialintermsof sizedistributionafterfragmentationandtheresultinginsert sizeofthegenomicPacBiolibrary.

5.AchievedbyshearingofDNA.CovarisS2orLE220System (500bp < 5000bp),Covarisg-Tubedevices(>6000bp)or HydroShearinstrument(>5000bp)mightbeused.

6.Nicks,abasicregions,thyminedimers,cytosinedeamination, blocked30 -ends,andoxidationdamagesitesaretreatedwith theuseofaDNA-damagerepairmix.

7.T4DNApolymeraseisutilizedforbothfillingin50 overhangs andtheremovalof30 overhangs.T4polynucleotidekinase phosphorylatesthe50 hydroxylgroup.Thus,bluntendsare formed.

8.Double-strandedDNAmatrixcappedbyhairpinloopsforms theSMRTbelltemplate(Fig. 2).Intermsoftopology, SMRTbelltemplateiscircular,whileregardingstructure,itis linear.TheligatedadaptersprotecttheendsoftheDNAfragmentsfromtheactionofexonucleases(exonucleaseIII,exonucleaseVII)utilizedfordegradationofthefailedligation productsoradapterdimersinthefollowingAMPure® PB beadspurificationsteps.

9.Thesequencingprimer-bindingsitesarelocatedwithinthe loopsoftheligatedhairpinadapters.Theprimershybridize tobothendsoftheSMRTbelltemplate.Higherstabilityofthe primersisassuredby20 -methoxymodifications.

10.ForbindingofDNApolymerasetotheprimer-annealed SMRTbelltemplates,incubationat30 Cfor30minis performed.

11.EfficacyofloadingSMRTbelltemplatesintoZMWsdepends ontheinsertsize;SMRTbelltemplatescontainingsmaller fragmentslinkmorepotentlythantheseincludinglarger inserts.Librarieslargerthan1000bpareimmobilizedinthe bottomofZMWsbyparamagneticbeads.TheMagBeadloadingprocedurestartsfromhybridizationbetweenthepoly-Atail ofthesequencingprimerandtheoligodTonthesurfaceof magneticbeads.Afterthewashingsteps,SMRTbell-MagBead sampleismovedtoa96-wellplate,loadedontheinstrument, andthenputintotheSMRTCellforimmobilization. TheMagBeadstationmovesthebeadsaroundthesurfaceof theSMRTCellinordertoimmobilizeSMRTbelltemplatesat thebottomoftheZMWs.

12.Specification >500Mbrawdataperpackage( 3%),polymerasereads >6000bp.

13.Read-length(>50),subread-length(>50),andreadquality (>0.75)filterswereimplemented.

14. catpacbio/*.fastq > pacbio.fastq

15. canugenomeSize¼4.9mmaxMemory¼20maxThreads¼12 minOverlapLength¼500-pacbio-rawpacbio.fastq

16.with.fofnreferringto“fileoffilenames”.

17.Thesearethemainoutputfilesgeneratedbytheprimary analysispipelineonPacBioRSII.

18. samtoolsfaidxcanu/canu.contigs.fasta

19. pbalign--nproc12--forQuiverpacbio.fofncanu/ canu.contigs.fastaquiver.cmp.h5

20. quiver-j12-rcanu/canu.contigs.fasta-oquiver. gff-oquiver.fastaquiver.cmp.h5

21. prokka--cpus12--compliant--centreXXX--rfam-force--outdirprokka_1/--prefixIFB_0099-genusDickeya--speciessolani--strainIFB_0099 genome.fasta

Acknowledgments

AllsequencingandcomparativegenomicsresearchtaskswereconductedthankstofoundingfromNationalScienceCentreinPoland via2014/14/M/NZ8/00501grantedtoE.L.A.M.P.issupportedfromNationalScienceCentreinPolandvia2016/21/N/ NZ1/02783.

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TheIlluminaSequencingProtocolandtheNovaSeq6000 System

AlessandraModi,StefaniaVai,DavidCaramelli,andMartinaLari

Abstract

TheNovaSeq6000isasequencingplatformfromIlluminathatenablesthesequencingofshortreadswith anoutputupto6Tb.TheNovaSeq6000usesthetypicalIlluminasequencingworkflowbasedonlibrary preparation,clustergenerationbyinsituamplification,andsequencingbysynthesis.Flexibilityisoneofthe majorfeaturesoftheNovaSeq6000.Severaltypesofsequencingkitscoupledwithdualflowcellmode enablehighscalabilityofsequencingoutputstomatchawiderangeofapplicationsfromcompletegenome sequencingtometagenomicsanalysis.Inthischapter,afterexplaininghowtoassembleanormalizedpool oflibrariesforsequencing,wewilldescribetheexperimentalstepsrequiredtorunthepoolsonthe NovaSeq6000platform.

Keywords Libraryqualitycontrol,Libraryquantification,Sequencingpool,Standardsequencing workflow,XPsequencingworkflow,Runsetup

1Introduction

TheNovaSeq6000Systemisaproduction-scalesequencingplatformfromIllumina,Inc.TheinstrumentmakesuseoftheIllumina sequencingbysynthesis(SBS)chemistryandenablesthemassively parallelsequencingofbillionsofDNAfragmentsintherangeof 50–500baseswithanoutputupto6Tb.Besidethehighthroughputandcost-effectivesequencing,theNovaSeq6000 offersflexibleoutputandrun-timeconfiguration.Multipleflow celltypessupportawideoutputrange.Usercanchoosebetween fourflowcelltypes(SP,S1,S2,andS4)anddifferentreadlengths toeasilyadjustoutputandsamplethroughputofsequencingrunto aspecificproject(Table 1).Moreflexibilityisachievedwithindividuallaneloading(Xpworkflow):twolanesforSP,S1,andS2flow cells;fourlanesforS4(Fig. 1).Additionally,theinstrumentcanrun oneortwoflowcellsofthesametypeatatime.Duetoscalability, theNovaSeq6000couldbeappliedinseveralapplicationsspanning fromcompletegenomeandexomesequencingtotarget

AlessioMengonietal.(eds.), BacterialPangenomics:MethodsandProtocols,MethodsinMolecularBiology,vol.2242, https://doi.org/10.1007/978-1-0716-1099-2_2, © SpringerScience+BusinessMedia,LLC,partofSpringerNature2021

Table1

NovaSeq6000flowcellsandoutputs

(Billionreads)

(h)

SP 2 50bp65–8020.6–0.81.3–1.613

SP 2 150bp 200–25025

SP 2 250bp 325–40038

S1 2 50bp134–16721.3–1.62.6–3.213

S1 2 100bp266–33319

S1 2 150bp400–50025

S2 2 50bp333–41723.3–4.16.6–8.216 S2 2 100bp667–83325

S2 2 150bp1000–125036

S4 2 100bp1600–200048–1010–2036 S4 2 150bp2400–300044

Fig.1 NovaSeq6000flowcelltypes.FromlefttorightSP,S1,S2,andS4flowcellrespectively.Lanessize andnumberaccordingtokitoutput

approaches,transcriptomeandgeneexpressionanalysis,andshotgunmetagenomics.

AsinotherIlluminaNGSsequencingsystems,theNovaSeq 6000workflowincludesfourbasicsteps:

1. Librarypreparation.Thefirststepistheconversionofthe genomicDNA(gDNA),cDNA,orPCRampliconsintoa sequencinglibrary.DNAfragmentsarecoupledwithtwooligonucleotidesadapters(namedasP5andP7)carryingspecific sequencesthatenablethesubsequentamplificationand sequencingstepsintheIlluminasequencingsystems.During librarypreparation,shortindividual“barcode”sequences (“indexes”)arealsoaddedtothestandardadaptersinorder toallowthesimultaneoussequencingofDNAfragmentsfrom differentsamples(multiplexing).

Twomaindifferentapproachescanbeappliedtoobtain sequencinglibrariesfromgenomicDNAorcDNA.Inthe “TruSeq”approach,asequencinglibraryisgeneratedbyrandommechanicalfragmentationoftheDNAsample(usually withanultrasonicator),followedby50 and30 adaptersligation [1].Alternatively,followingthe“Nextera”workflow,fragmentationandligationreactionscanbecombinedintoasinglestep employingatransposon/transposase-mediatedcleavagemechanism(“tagmentation”)[1].Genomicfragmentsaresubsequentlyamplifiedusingprimerstargetedtoadaptor sequences.Duringthelibraryamplificationstep,individual indexesareincorporatedintotheadaptorsofeachsampleby meansofsample-specificindexingprimers;bothsingleordoubleindexingcanbeperformedinordertoincreasemultiplexing.Severalcommerciallibrarypreparationkitsareavailable Mostofthecommercialkitshavebeenoptimizedforspecific applications(e.g.,transcriptomics,epigenomics,ChIP-seq, PCR-freelibrary).Alternatively,customin-houseprotocols havebeendeveloped[2, 3].Whendegradedmaterialisanalyzed,asinthecaseofDNAextractedfromancientbiological samples,fragmentationstepisusuallyskipped,andadaptersare ligateddirectlytotheendsofeachDNAmolecules.Amplicons targetedsequencing,asinatypicalmicrobialmetagenomic16S analysis,canbesimplyachievedbyusinguser-definedPCR primerswithoverhangadapters;asubsequentlimited-cycle amplificationstepisperformedtoaddmultiplexingindexes andIlluminasequencingadapters[4].Forsomeapplications wherehugenumbersoftargetsarerequired,specificamplicon panelscanbedesignedandpurchased.

Afterqualitycontrolandquantification,indexedlibraries arenormalized,pooledintherequiredmolarity,andthen loadedintothesequencingplatformalongwithbuffersand reagents.

Despitetheoriginalsourcematerialand/orthespecific librarypreparationprotocolfollowed,eachtypeoflibrarycan bevirtuallysequencedinalltheIlluminasequencingplatforms. Thechoiceofaspecificsequencerandoftheappropriate sequencingkitisthereforemainlydrivenbytherequiredoutputandcost-effectiveness.

2. Clustergeneration.Forclustergeneration,thelibraryisloaded intoaflowcell,aglassslidecontainingsmallfluidicchannels andalawnofsurface-boundoligonucleotidescomplementary tothelibraryadapters.TheDNAlibraryhybridizetothese oligonucleotides,temporarilyimmobilizingindividualDNA fragmentsontotheflowcell.Afterlibraryhybridization,polymerases,dNTPs,andbuffersarepumpedintheflowcell.Each singlefragmentisthencopiedseveralthousandtimestogeneratedistinct,clonalclustersthroughanin-situamplification processknownas“bridgePCR”[5].Whenclustergeneration iscomplete,thetemplatesarereadyforsequencing.

TheNovaSeq6000makesuseofpatternedflowcellsthat containbillionsofnanowellsatfixedlocationsacrossboth surfacesoftheflowcell[6].Thestructuredorganizationprovidesfixedspacingofsequencingclusters,makingtheflowcells lesssusceptibletooverloading,andreducesruntime(noneed tomapclustersitesduringsequencing).Additionally,higher clusterdensityleadstomoreusabledataperflowcell.

3. Sequencing.Withineachcluster,templatesaresequencedby meansofIlluminasequencingbysynthesis(SBS)chemistry. TheSBSchemistryisareversibleterminator–basedmethod usingasinglebaseextensionandcompetitiveadditionof nucleotides[5].Asallfournucleotidesarepresentduring eachsequencingcycle,themethodminimizesincorporation biasandsignificantlyreduceserrorsandmissedcallsassociated withstringsofrepeatednucleotides.Illuminasequencingkits aredesignedtosupportbothsingle-endandpaired-end sequencing.Inpaired-endsequencingtheDNAfragmentsof eachclusterarefirstsequencedfromoneend(asinthesingleendsequencing)andthenfromtheoppositeend.This approachgenerateshigh-qualitysequencedataimproving alignmentandgenomeassembly.Additionally,paired-end sequencingcanhelpinidentifyingauthenticreadsfromendogenousDNAwhenlibrariesfromancientanddegradedsamples areanalyzed.

Duringeachsequencingcycle,asingleflorescentlabeled deoxynucleotidetriphosphate(dNTPs)isaddedtothenucleic acidchains.Thelabeldyesactasareversible30 blockersand serveaterminatorforDNAstrandsynthesis.AseachdNTPis added,thefluorescentlydyeisimagedandthencleavedto allowincorporationofthenextnucleotide.Ateachsequencing cycle,fluorescentemissionfromeachclusterisregisteredand normalized.Emissionwavelengthandintensityarethenused bycontrolsoftwareoftheinstrumenttocallthebases.For nucleotidedetection,theNovaSeq6000makesuseofthe two-channelSBSmethodthatrequiresonlytwoimagesper cycle,insteadoffour,andacceleratessequencinganddata

processingtimes.Ratherthanfourseparatedyes,two-channel SBSusesamixofdyes.ImagesaretakenofeachDNAcluster usingonlytwodifferentwavelengthfilterbands.Clustersseen inredorgreenimagesareinterpretedasCandTbases, respectively.Clustersobservedinbothredandgreenimages (yellowclusters)arerecognizedasAbases,whileunlabeled clusterscorrespondtoGbases.TheNovaSeq6000usually produces75–85%ofsequencedbasesover30qualityscore, thatmeans0.1%probabilitythatabasewascalledincorrectly.

4. DataAnalysis.Afterasequencingruniscompleted,dataare availablefordownstreamanalysis.Inordertoassignthereads toeachdifferentsample,rawdataarefirst“demultiplexd”by meansofindexesrecognition.Thesequencedreadsarethen mappedtoareferencegenomeorsequenceandanalyzed accordingtospecificapproaches(e.g.,SNPsandindelidentification,phylogeneticormetagenomicanalysis).

Whilesteps1(LibraryPreparation)and4(Dataanalysis) canbecarriedoutinanymolecularbiologylaboratoryor computationfacilityregardlessofthesequencingmachine andtherunschedule,steps2(ClusterGeneration)and 3(Sequencing)arephysicallyexecutedintothesequencing platformduringtherun.

Inthischapter,wewilldescribetheexperimentalsteps requiredtorunasetoflibrariesontheNovaSeq6000sequencingplatform(Fig. 2).

Wewillfirstprovidesometipsforthefollowing:

(a)Toperformlibraryquantificationandqualitycontrol.

(b)Tonormalizeconcentrationandprepareanequimolar pooloflibraries.

Then,accordingtothespecificmanualsofinstructionbyIllumina[7],wewillreporttheworkflowthatshouldbefollowedfor thefollowingsteps:

(a)Topreparereagentsforclustergenerationandsequencing.

(b)Toloadthepoolsoflibrariesandreagentsontheflowcellwith bothstandardandXPworkflow.

(c)Tosettheinstrumentfortherun.

2Materials

2.1General Equipmentand Reagents Automatedelectrophoresissystemwithrelatedreagentsanddata processingsoftware. Fluorometerwithrelatedreagents. Vortex.

2.2Proprietary Reagentsand Materialsfrom Illumina1

Fig.2 Schematicoverviewoftheexperimentalstepsforpoolingsequencing librariesandrunthepoolsontheNovaSeq6000

Microspin.

Micropipetteswithdisposabletips. Icebath.

10mMTris–HCl,pH8.5. 0.2NNaOH.

ForlibrarydilutionsandpoolassemblingwesuggestusingLoBind orsiliconizedtubes.

PhiXControlv3(FC-110-3001).

NovaSeqReagentKits:severalkitsareavailable,accordingto differentflowcellsandoutputs(Table 1).Eachkitprovidesthe following:

1 Atthetimeofwriting,theauthorsarenotawareofalternativesuppliersofferingsequencingreagentsand materialscompatiblewithIlluminaNovaSeq6000System.

Another random document with no related content on Scribd:

The Project Gutenberg eBook of Shuddering castle

This ebook is for the use of anyone anywhere in the United States and most other parts of the world at no cost and with almost no restrictions whatsoever. You may copy it, give it away or re-use it under the terms of the Project Gutenberg License included with this ebook or online at www.gutenberg.org. If you are not located in the United States, you will have to check the laws of the country where you are located before using this eBook.

Title: Shuddering castle

Release date: October 4, 2023 [eBook #71806] Most recently updated: October 18, 2023

Language: English

Original publication: New York, NY: Green Circle Books, 1936

Credits: Greg Weeks, Mary Meehan and the Online Distributed Proofreading Team at https://www.pgdp.net (This book was produced from images made available by the HathiTrust Digital Library.) *** START OF THE PROJECT GUTENBERG EBOOK SHUDDERING CASTLE ***

SHUDDERING CASTLE

BOOKS

NEW YORK

COPYRIGHT, 1936

[Transcriber's Note: Extensive research did not uncover any evidence that the U.S. copyright on this publication was renewed.]

Printed in the United States of America

SHUDDERING CASTLE

S C is not a mystery novel in the generally accepted sense. It is a novel with a mystery; a highly imaginative story, with revelations in the field of radio and short-wave broadcasting. None of the strange events recorded oversteps the boundaries of accepted natural laws.

In this novel of exciting action, radio communication is established between Earth and Mars, with a world-girdling hook-up from Radio Center, in New York City, and the reader will be amazed to find that the Martians are human beings like ourselves, subject to the same laws, the same temptations and passions which affect humanity. Into this pulsating picture of tensed American life of the near future, comes another revelation from the sky. This brings the reader to the drama of a frightening but plausible visitor from the jungles of Mars to this world, whose presence in the old spooky castle of an eccentric millionaire-scientist on Long Island causes great fear to its inmates when night falls.

But there is thrilling romance to warm your hearts, the infatuation of a young newspaperman for the alluring debutante niece of the old scientist; a humanly drawn boy and girl who are caught in the violent web of mystery and sudden death. S C is a unique study in the mysterious recesses of the universe.

SHUDDERING CASTLE

IAs a staid and wealthy New York family, of distinguished but remote English ancestry, we moved formally and rather arrogantly within our small, exclusive circle, holding on grimly to the traditions and elegancies of the past. During the winter season, we viewed the outside world placidly, and with the respectful composure of middleage, from the dignified privacy of our red brick mansion in Washington Square.

On May first, as regular as clockwork, year in and year out, and with all the solemnity of a ritual, we put our elaborate upholstered furniture in linen shrouds, veiled the somber, scowling family portraits in their dull gold frames with fly-netting, boarded up the windows and doors, and went to the country. Our summer home is called The Castle, and it is situated at Sands Cliff, Long Island. As a family we resembled nothing so much as this embattled stone fortress, of oldworld design, in which we spent more than half the year.

As long back as I can remember, we had successfully preserved the family's seclusion from the living world. Wherever we happened to be, in town or country, we had protected our privacy with shuttered windows, and massive iron gates that were secured both day and night with heavy chains. Numerous signs of "Private" and "No Trespassing Allowed" dotted our grounds like grave markers.

And then, quite suddenly, our lives became incredibly transformed. A series of weird events brought us out of our privacy and seclusion— brought us plenty of excitement and trouble and even horror.

But that was not to be wondered at, with Henry, my elder brother, suddenly developing a mania for research in scientific matters, especially the science of heavenly bodies and the phenomena of radio. He did not pretend to be a scholar, although he had cultivated scholarly habits most of his life. Inexplicably, this mania had seized him late in life; a sort of bursting out of the abnormal repression which held us all in thrall, no doubt as the result of our long seclusion from the outside world and following the drab and barren routine of our lives with such punctilious rigidity

Ample means had enabled him to completely outfit an observatory, with a powerful telescope, at our summer residence. Here he would spend hours gazing into the abyss of space. He saw things up there the trained, professional astronomer never saw, or ever hoped to see —colliding suns, formation of temporary stars, the rejuvenescence of dying worlds, and gaseous explosions in the Milky Way.

One of his pet theories was that the planet Mars was inhabited by a race of people like ourselves, and that their men of science had long been trying to establish radio communication with the earth. The static on our radio set which annoyed me intensely, would galvanize Henry with delight and hope, and his eyes would glisten almost frenziedly behind their horn-rimmed spectacles.

"Those are distinctly electro-magnetic waves," he would say, "that come from some point far off in space, and they are not due to any terrestrial disturbance like thunderstorms, local or distant."

There was no opening, no escape, from Henry once he got started on the galactic radio waves as differing from the cosmic rays and from the phenomenon of cosmic radiation.

"I'm telling you, Livingston," he once declared in an excited, highpitched voice, "that man has only begun his conquest of time and space. There are no limitations to human achievement. The world is on the threshold of things unheard of, undreamed of. I have no doubt that we will soon be able to establish radio communication with Mars, and with my leisure, money and the required taste for science, I feel that I am admirably fitted to make it come true."

And from that day he was changed, secretive. He refused to tell me what he had discovered. Again and again I begged him to explain and always it was the same vague answer, the same shake of the head, and tightened lips.

It all seemed fantastic and visionary then, Henry's theories about Mars and interstellar communication, but when unusual things began to happen and our peaceful and ordered living was suddenly and violently disturbed, I realized, as never before, that visions often come to reality in an unbelievable way.

At the time we were thrown into such turmoil, and the dread spotlight of publicity centered upon us, our family consisted of Henry and myself, both bachelors; Jane, our spinster sister, and Patricia Royce Preston—Pat for short—a very fascinating young person, who had come to live with us at the tender age of fourteen, after the shocking death of her parents, our youngish sister, Virginia Royce Preston, and her husband, Allston, who were killed in an air-liner crash near Paris.

There is something strangely lovable about a young girl in the process of growing up. The advent of Pat meant, of course, less privacy and the trampling down of staid personal habits and family customs which we held virtually sacred. The fact that we were old and queer and our household drab and rather grotesque, in comparison to the modernistic and rather barbaric splendor of our more fashionable friends, scarcely troubled her. Nothing seemed to matter but that this bright-eyed, brown-haired girl should concentrate all her love and devotion on a trio of old fossils. A warm affection grew between us and our pretty niece. As she blossomed into young womanhood our lives became centered in her. She was now eighteen.

Although we were born rich, and received a huge income from the heritage of vast and various real estate holdings on Manhattan Island, both Henry and myself, strangely enough, had never splurged, and never married. I am sure the thought of matrimony never entered Jane's mind. Our natural emotions seemed to be stirred and exalted only by the importance of our family name and our wealth. Romantically, we were strangely neutral, as though, in the pursuit of riches, the family stock had been sort of washed out.

After our college days, Henry and I grew into old-fashioned, mellow bachelorhood, aloof from the world and very self-sufficient, and glad to have it so. Henry had just observed his sixty-fifth birthday when our lives became so tempestuous and convulsed. I was two years his junior. Jane had just turned sixty. As progeny, we seemed to have come into this world in swift successiveness, as though the marriage of our revered parents had fulfilled its promise in a bunch.

For an entire summer Henry lived virtually in seclusion in his observatory without any tangible result. Sweeping the sky with his telescope for anything that might happen. But nothing transpired. Yet he persisted. Finally, he detected a tiny comet, apparently on its way to the earth. At first it appeared no larger than a pin-prick of light, with a small, meteoric tail.

The night he made the discovery, he got me out of bed to see it, but I was in no mood or condition for sky gazing. In addition, looking into the eye-piece of the telescope made me a little sick and dizzy. I couldn't see a thing. Deciding that he was suffering from a delusion, I went back to bed.

The odd thing was that Henry was right. He had actually witnessed the phenomenon of impact of two small planets which produced the comet. As he explained it afterwards to a group of eminent scientists, this collision of two celestial bodies had produced a distinct flash of light, out of which had grown a spiral swarm of very brilliant particles, and he had watched them as they took on orbital motion.

The comet soon became the most impressive and magnificent sight I have ever seen, stretching its scimitar-like form half across the heavens. Its wonder and beauty dragged New Yorkers up in the small hours, to gaze at it with fascinating awe. Many regarded it with terror, others with superstitious dread. In churches throughout the land, the people prayed: "Lord save us from the devil, and Royce's comet!"

The comet was not only named after Henry but his discovery was acclaimed by scientists the world over, and he was chosen a fellow of the two leading scientific bodies of America and England. While still rated as an amateur in science, nevertheless, many learned men began to look upon him as the depository of authority and authenticity in matters relating to the mysteries of the solar system.

Having disclosed something to the world in the order of creation, Henry became imbued with an overpowering sense of his own importance as a man of science; his ambitions soared to unsurmountable heights. The discovery of the comet having been far easier than he had dared dream, he now turned with profound

intentness to establish radio communication with Mars. He began talking in a familiar and chatty way about the people on Mars, and to hear him talk one would think that he was going there for a week-end of golf.

In this project, he had enlisted the able assistance of Serge Olinski, assistant research engineer of the National Radio Corporation, whose unexceptional qualifications included an honor degree in cosmic ray research, with distinction in astronomy. Their experimental activities, in trying to pick up and decode the galactic radio waves, which both believed constituted some kind of interstellar signaling, were carried on behind locked doors, either at Henry's observatory in the country, or in Olinski's laboratory in the NRC Building, in the new Radio Center Annex.

Olinski was a queer shrinking soul, and any sort of publicity to Henry was equally distasteful. They were two of a kind, in this respect. Notwithstanding all the praise and attention given to Henry by the press during the comet furore, he treated reporters with the utmost contempt, and accused them of being dishonest rogues. One reporter in particular he hated and feared. Just mention to him the name of Robert McGinity of the New York Daily Recorder, and his correctly chiselled and aristocratic features would crinkle up in rage and horrible chuckles would issue from his thin lips like unnamable profanities.

He had never forgotten his first encounter with McGinity on the telephone, nor had he ever forgiven the reporter for what he called an utterly disreputable transaction in news. But the business of reporting is at least an honorable one, and reporters have to get their stories, somehow.

This fellow, McGinity, published the first report of Henry's discovery of the comet, and scored a beat by calling him up and giving the impression that he was one of the assistant astronomers at Harvard University I had no suspicions then how the information had trickled into the office of the Daily Recorder, but I believe now that our solemn-visaged butler, Orkins, who afterwards turned out to be so

mercenary and treacherous, tipped off this morning paper, which paid liberally for exclusive stories.

It was the night following Henry's detection of the comet when he was aroused out of a sound sleep to answer an important telephone call. If I hadn't been up and overheard the conversation, I wouldn't have believed it possible for any man to be so easily deceived. But gullibility is one of Henry's weaknesses. I switched into the conversation from an extension on the second floor.

Henry seemed to have some recollection of the name of the Harvard professor, as it came over the telephone, and at first was a little taken aback and curious that the news of his discovery should have become known. Despite this, he told all about his detection of the new comet, and proudly, omitting no detail. It would have been ungrateful on his part to have distrusted the man at the other end of the wire, after he had gone to the trouble and expense of calling up, obviously from Boston, and it seemed so unlikely that any one outside astronomical circles would be interested in the discovery. Up to that time, Henry had had no dealings with reporters. By exercising extraordinary discretion, he had managed all his life to keep out of the news, except for occasional real estate transactions, and had always avoided any encounter with the press.

After he had answered heaps and heaps of questions, the voice at the other end said: "Thanks, Mr. Royce. Thanks a lot. Darned good of you to tell me all this."

An oppressive silence descended. By that time, Henry must have guessed that he had been gulled. I got his voice but I missed the play of expression on his face.

"Who is this speaking?" he asked again. "Who the devil are you?"

"Bob McGinity of the Daily Recorder," came the prompt reply.

Henry gave a nervous jump. "What?" he gasped angrily. It was evident that he was utterly taken by surprise. "I—I find your action in calling me up quite incomprehensible, Mr. McGinity. I imagined that —that—"

"Pardon me," the reporter retorted with some dignity "I never said I was an assistant professor of astronomy at Harvard. I simply asked if you knew of such a person, and you said you did, and then you proceeded to tell me exactly what I wanted to know."

"But surely you're not going to publish this," Henry fumed. "It's too immature. You must keep it out of the newspaper."

"I'm sorry but I have no power to do so, Mr. Royce," the reporter replied. "And no inclination, Mr. Royce."

Henry clawed at the telephone instrument with trembling fingers. "If I had you here, young man," he shouted, "I'd break your damned neck."

He hung up with a bang, and I don't think he slept a wink the rest of the night. And it was entirely due to this experience that he and Olinski took every precaution that nothing should leak out concerning their research in interstellar signaling, which, as far as I could learn, at the time, had entered on the final and exciting stage of their experimental work.

Henry's actions indicated that his mind was still working feverishly on this subject; he even raved about it in his sleep, according to his Filipino valet, Niki. But about his and Olinski's doings, not a word to me. When I would ask him if they had found anything worth finding, he would reply: "Just you wait, and see;" a vague term which he refused to make more definite.

In the silent watches of the night, he would sit at his telescope, his eyes trained on that beautiful, reddish planet, Mars. One morning, at four o'clock, I found him there, clad only in his pajamas, and he strongly resented my intrusion. But I had a task to perform, and that was to see that he got his proper rest. I had no wish that any member of our family should become psychopathic.

"Henry!" I exclaimed, rather harshly; "you've only a few hours before breakfast-time. Go to bed and get a bit of sleep."

I think he realized, instinctively, that I was not in sympathy with this business of trying to pick up radio signals from Mars. It all seemed so useless and incredible. His secret experiments had been in

progress now for about a year The tumult aroused by the discovery of the comet seemed a thing long past and forgotten. The memory of the public is short. Newer sensations had taken its place.

In this latest mad, scientific quest, Henry reminded me of one of Jane's goldfish, which swims in its bowl, and swims and swims, thousands of miles, perhaps, and then finds itself a few inches from its starting point. So one day I resolved to bring the matter to an issue. I slipped into his room just after he had disrobed and donned a dressing-gown, preparatory to taking a bath and dressing for dinner.

"Henry," I began, rather abruptly, "study and action are worth while, only when they lead you some place." But I was not destined to finish what was in my mind to say.

"I beg pardon, Livingston, if I disturb you," he interrupted in his meekest accents, and then went into his bathroom, and closed the door.

Determined to have my say, I followed him to the door, and knocked. The door opened, and his face, meek and anxious, looked out at me through a narrow crack.

"Henry!" I implored. "If I could only see you for a few minutes—" "No!" he said, and shut the door. A second later, I heard the bar shoved into its slot.

There was nothing unusual in Henry locking himself in his bathroom, for he had the distressful habit of sitting in his bath-tub, by the hour, smoking and thinking. His bathroom seemed to be the only quiet retreat in the castle which afforded the complete solitude and privacy necessary for the employment of his brain cells. He felt that here he could relax, just as Napoleon did, after undue fatigue, dictating letters and giving important military orders from his steaming bathtub.

I have often wondered where Sir Isaac Newton was sitting, at his home in Woolsthorpe, England, when the fall of an apple, so legend tells us, suggested the most magnificent of his discoveries, the law of universal gravitation. There is no evidence to refute that he was

sitting in one of those queer, early English bath-tubs, looking out of the bathroom window, at his apple orchard.

I never see Rodin's famous sculpture, "The Thinker," but I am reminded of Henry, sitting in his bath-tub, thinking and thinking, especially during the early part of the eventful summer of which I write.

Evidently some fresh idea had come to him while in his bath on the evening I persisted in assailing his peace of mind. With startling suddenness he donned his bath-robe, rushed to the telephone, and communicated with Olinski. As quickly as possible, the next day, they got to work on Henry's idea. Then problems began to straighten themselves out. As to what they had discovered, they said nothing at the moment.

Soon after, however, an avalanche of adventure, mystery and excitement came thunderously down upon us, throwing our lives into chaos.

II

As I begin my narrative, my mind travels back for a moment to the days of my youth, and I am made more vividly aware of the changes that have taken place in the world. We are living in a new era now— a period marked by a series of strange occurrences, manifestations of the weird powers that lurk in outer space. The New Deal has passed into history. A strangely remote time ago, that was....

The laboratory has supplied us with the basic means of lifting the curtain of space from scenes and activities at a distance. A system of sight transmission and reception, comparable in coverage and service to the world-wide hook-up of sound broadcasting, has brought all nations closer together. In the friendly exchange of ideas and feelings through the medium of television and the radio, the whole civilized world enjoys common participation.

Nationalism no longer endangers the peace of the world. All war debts between nations have been settled, and tariff barriers laid low. Internationalism reigns supreme, to the spirit and benefits of which Henry contributed his share by engaging servants representing seven nationalities. Thus we harbored at the castle of Sands Cliff about every conceivable question of society, politics and religion.

Our summer castle is such a place as you read of, in romances of the Middle Ages. It was built more than half a century ago by a wealthy New York society woman who must have had a strain of poetic romanticism in her veins. When Henry purchased the place, it was almost in ruins.

It is perched on the summit of a precipitous sand cliff, commanding an excellent view of Long Island Sound. From its windows, on a bright day, the majestic towers of New York appear dimly etched against a mauve horizon like the spires of a magical city. There it stands, dark and foreboding, and ivy-clad, in its own grounds, surrounded by a high brick wall. The main entrance gate is approached by a dark avenue which winds through a heavily wooded park. There is no other dwelling within a mile.

There are many mullioned windows in its slim, peaked towers. Inside, a clutter of rooms—endless rooms—some of them in the upper floors unused and smelling dusty and dank. The front door opens on a brick terrace, which has a stone balustrade as a protective measure against a sheer drop of two hundred feet to the rocky base of the cliff. From the east end of the terrace, stone steps wind down to a private yacht landing and a long stretch of beach, fenced in with barbed wire.

An outstanding feature of the castle is its galleried entrance hall, with its darkly gleaming oak panelling and great, stone staircase; a hall so large that when one speaks, the sound is echoed like the whispering of ghosts from the high, oak-timbered ceiling.

There is a queer element of solitude and uncanniness that always cloaks the castle at the twilight hour, before Orkins, in his routine of duty, switches on the lights. I noticed it particularly, one summer evening, about the middle of August, as I walked up and down the

terrace, dinner-jacketed and smoking, awaiting the arrival of our two dinner guests, Serge Olinski and His Highness Prince Dmitri Matani.

The sun had gone down in a cloudless, violet sky, and purplish twilight had settled on the Sound and the marshland, stretching westward to a cove, where the lights of the village of Sands Cliff were beginning to twinkle. The silence was more oppressive than the heat. Now and then it was broken by a distant tugboat whistle, like the hoarse croak of a frog, and the faint calling of a thrush for its mate in the thick shrubberies that fringed Jane's flower garden, on the north side of the castle.

Far out in the Sound, two sail-boats were drifting along like tired ghosts. Presently the fringe of the opposite shore became magically outlined by tiny strands of lights. As the gloom of night slowly enveloped the scene, an island lighthouse, a mile away, began to flash its beacon over the dark, graying water with clock-like regularity.

Against this flashing light, the ruins of our own lighthouse showed dark and jagged, on a small, rocky island, rising out of the Sound about a quarter of a mile off our shore, and within easy rowing distance from the yacht landing. Henry had recently purchased the island from the Government, and it was now a part of our Sands Cliff estate. The old beacon tower of stone was built in 1800. In oilburning days, its light had counted for something, but now it was nothing but a picturesque ruin, and largely populated during the summer by bats.

I had no sooner turned my gaze on the ruined lighthouse when a big bat swooped down at me out of the darkness. Only the night before, one of them had got into my bedroom. I've never been able to overcome my early fear of these nocturnal flying mammals. To my childish imagination, they were the very spirits of evil. I was in no mood this night to be pestered by them. A vague uneasiness possessed me, an uneasiness caused on one hand by Henry's strained and haggard look, and on the other, by his encouraging Prince Matani's attentions to Pat.

Perhaps at the moment, his crazy quest in interstellar communication annoyed me most. I had already suggested to Jane that we send him to a psychoanalyst to be overhauled. This delving into the unknown was too ponderable a matter for a man of his years. It had become fixed on his mind with all the power of an obsession. All that day he had not stirred from his observatory, and now Olinski was coming from town to give a verbal report of his own findings. Much cogitation, much secrecy was, in effect, nothing at all. Unless they now had found the key. Was it possible that Olinski might be bringing a transcribed cipher of a radio message from Mars? His eager acceptance of the invitation to dinner seemed to hold an important significance for Henry.

Desperately bothered by both problems which confronted me, the bats made things more annoying still. Then, sudden-like, in the haunting stillness, I saw something moving towards me from the blackish void of trees and shrubbery bordering the west end of the terrace. At first, I was conscious only of an oncoming shadow, advancing with a rapid, noiseless movement.

I could feel my pulse jumping. Whoever or whatever it was, there was a risk. Rather than face the risk, I moved quietly but swiftly across the terrace towards the front door. But that did not stop the oncoming something; it had suddenly changed its direction and was coming right at me.

Luckily at that moment, the lights were turned on in the lower part of the castle. Then Orkins opened the front door, and gave voice to a surprised exclamation as he saw me making hurriedly for the doorway.

Suddenly I stopped, and turned. The glow of a floor lamp in the entrance hall had spread fanwise across the terrace, and into this arc of light strode—Serge Olinski.

"Oh, hello, Olinski!" I exclaimed, with respectful familiarity, and very cordially, stretching out my hand, and smiling to myself at the start he had given me, coming like an abortive something out of the shadows of the terrace. "That you?"

"Yes; it is I," Olinski replied, shaking my proferred hand, and breathing rather heavily.

I faced a short, dumpy, middle-aged man, with a paunch, and a Russian cast of countenance. Small, intelligent black eyes gleamed through shell-rimmed glasses, from a round face fringed with a short, black beard. He carried his hat, and I observed that his primly sleeked hair was as black as his beard. I had a suspicion that he dyed them.

"I caught an early train from the city, in order to enjoy the benefit of a walk from the village to your beautiful castle," he explained, half breathlessly, "after a most exacting but successful day in the laboratory. A million apologies if I have delayed your dinner."

"Time is infinite in the country, especially on a fine night like this," I remarked lightly, as we entered the hall, and Orkins relieved him of his black top-coat and hat. His dinner jacket, I noticed, was much too small for him, and his waistcoat so short that it came perilously near revealing a section of his middle-age bulge. There were soup stains on his shirt-front, which indicated that his shirt had been out to dinner before.

As I waved him to a chair, I said: "You're really very punctual, even if you avoided our car which was sent to the station to meet you, and walked here. You can depend upon it, Prince Matani will not miss the chance to drive to the castle in state when he steps off the train."

Unconsciously my lips sneered as I spoke the young princeling's name. Olinski nodded and smiled understandingly "Ah!" he said. "I take it that you do not look with favor on the match your scholarly brother is about to arrange between your charming niece and my noble countryman?"

"To be frank, no," I replied.

"So I gathered. And why?"

"I have very strong reasons for opposing their marriage," I said; "and my sister, Jane, is just as dead set against it as I am. Every one knows that the Prince came to America to make a rich and advantageous marriage. Pat will soon come into a large inheritance

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