Hybridization Capture Using RAD Probes (hyRAD), a New Tool for … · 2016. 3. 22. · 3 40 Introduction 41 42 With the advent of next-generation sequencing, conducting genomic-scale

1

HybridizationCaptureUsingRADProbes1

(hyRAD),aNewToolforPerformingGenomic2

AnalysesonCollectionSpecimens 3

4

TomaszSuchan1‡*,CamillePitteloud1‡,NadezhdaS.Gerasimova2,3,AnnaKostikova3,Sarah5

Schmid1,NilsArrigo1,MilaPajkovic1,MichałRonikier4,NadirAlvarez1*6

7

1DepartmentofEcologyandEvolution,UniversityofLausanne,Lausanne,Switzerland,8

2BiologyFaculty,LomonosovMoscowStateUniversity,Moscow,Russia,9

3InsideDNALtd.,London,UnitedKingdom,10

4InstituteofBotany,PolishAcademyofSciences,Kraków,Poland11

‡Theseauthorsarejointco-firstauthorsonthiswork.12

*[email protected](TS);[email protected](N.Alvarez)13

14

15

.CC-BY 4.0 International licenseavailable under anot certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (which wasthis version posted March 22, 2016. ; https://doi.org/10.1101/025551doi: bioRxiv preprint

https://doi.org/10.1101/025551

http://creativecommons.org/licenses/by/4.0/

2

Intherecentyears,manyprotocolsaimedatreproduciblysequencingreduced-genomesubsetsin16

non-modelorganismshavebeenpublished.Amongthem,RAD-sequencingisoneofthemost17

widelyused.ItreliesondigestingDNAwithspecificrestrictionenzymesandperformingsize18

selectionontheresultingfragments.Despiteitsacknowledgedutility,thismethodisoflimiteduse19

withdegradedDNAsamples,suchasthoseisolatedfrommuseumspecimens,asthesesamples20

arelesslikelytoharborfragmentslongenoughtocomprisetworestrictionsitesmakingpossible21

ligationoftheadaptersequences(inthecaseofdouble-digestRAD)orperformingsizeselection22

oftheresultingfragments(inthecaseofsingle-digestRAD).Here,weaddresstheselimitationsby23

presentinganovelmethodcalledhybridizationRAD(hyRAD).Inthisapproach,biotinylatedRAD24

fragments,coveringarandomfractionofthegenome,areusedasbaitsforcapturinghomologous25

fragmentsfromgenomicshotgunsequencinglibraries.Thissimpleandcost-effectiveapproach26

allowssequencingoforthologouslocievenfromhighlydegradedDNAsamples,openingnew27

avenuesofresearchinthefieldofmuseumgenomics.Notrelyingontherestrictionsitepresence,28

itimprovesamong-samplelocicoverage.Inatrialstudy,hyRADallowedustoobtainalargesetof29

orthologouslocifromfreshandmuseumsamplesfromanon-modelbutterflyspecies,withahigh30

proportionofsinglenucleotidepolymorphismspresentinalleightanalyzedspecimens,including31

58-year-oldmuseumsamples.Theutilityofthemethodwasfurthervalidatedusing49museum32

andfreshsamplesofaPalearcticgrasshopperspeciesforwhichthespatialgeneticstructurewas33

previouslyassessedusingmtDNAamplicons.Theapplicationofthemethodiseventually34

discussedinawidercontext.Asitdoesnotrelyontherestrictionsitepresence,itisthereforenot35

sensitivetoamong-samplelocipolymorphismsintherestrictionsitesthatusuallycausesloci36

dropout.ThisshouldenabletheapplicationofhyRADtoanalysesatbroaderevolutionaryscales.37

38

39



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Introduction40

41Withtheadventofnext-generationsequencing,conductinggenomic-scalestudieson42

non-modelspecieshasbecomeareality[1].Thecostofgenomesequencinghassubstantially43

droppedoverthelastdecadeandrepositoriesnowencompassanincredibleamountofgenomic44

data,whichhasopenedavenuesfortheemergingfieldofecologicalgenomics.However,when45

workingatthepopulationlevel—atleastineukaryotes—sequencingwholegenomesstilllies46

beyondthecapacitiesofmostlaboratories,andanumberoftechniquestargetingasubsetofthe47

genomehavebeendeveloped[2,3].Amongthemostpopularareapproachesrelyingon48

hybridizationcaptureofexome[4]orconservedfragmentsofthegenome[5],RNAsequencing49

(RNAseq[6]),andRestriction-Associated-DNAsequencing(RADseq[7,8]).Thelatterhasbeen50

developedinmanydifferentversions,butgenerallyreliesonspecificenzymaticdigestionand51

furtherselectionofarangeofDNAfragmentsizes.RAD-sequencinghasprovedtobeacost-and52

time-effectivemethodofSNP(singlenucleotidepolymorphisms)discovery,andcurrently53

representsthebesttoolavailabletotacklequestionsinthefieldofmolecularecology.Thewide54

utilityofRAD-sequencinginecological,phylogeneticandphylogeographicstudiesishowever55

limitedbytwomainfactors:i)thequalityofthestartinggenomicDNA;ii)thedegreeof56

divergenceamongthestudiedspecimens,thattranslatesintoDNAsequencepolymorphismat57

therestrictionsitestargetedbytheRADprotocols.58

SequencepolymorphismattheDNArestrictionsitecausesaprogressivelossofshared59

restrictionsitesamongdivergingcladesandresultsinnullallelesforwhichsequencedata60

cannotbeobtained.Thislimitationcriticallyreducesthenumberoforthologouslocithatcanbe61

surveyedacrossthecompletesetofanalyzedspecimensandleadstobiasedgeneticdiversity62

estimates[9-11].Thisphenomenon,combinedwithothertechnicalissues–suchaspolymerase63

chainreaction(PCR)competitioneffects–isaseriouslimitationofmostclassicRAD-sequencing64

protocolsthatneedstobeaddressed.65



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Inaddition,RAD-sequencingprotocolsrelyonrelativelyhighmolecularweightDNA66

(especiallyfortheddRADprotocol[12]),notablybecauseenzymedigestionandsizeselectionof67

resultingfragmentsarethekeystepsforretrievingsequencedataacrossorthologousloci.68

Therefore,RAD-sequencingcannotbeappliedtodegradedDNAsamples,alimitationalso69

sharedbyclassicalgenotypingmethodsandampliconsequencing.Museumcollections,70

althoughencompassingsamplescoveringlargespatialareasandbroadtemporalscales,have71

notnecessarilyensuredoptimalconditionsforDNApreservation.Asaresult,manymuseum72

specimensyieldhighlyfragmentedDNA–evenforrelativelyrecentlycollectedsamples[13-73

15],limitingtheiruseformolecularecology,conservationgenetics,phylogeographicand74

phylogeneticstudies[16,17].Acost-effectiveandwidelyappliedapproachforgenomic75

analysesonmuseumspecimenswouldallowexploringoftenuniquebiologicalcollections,e.g.,76

encompassingrareornowextincttaxa/lineagesororganismsoccurringephemerallyinnatural77

habitatsandposingproblemsforsampling.Itwouldalsoallowstudyingtemporalshiftsin78

geneticdiversityusinghistoricalcollections,nowappliedonlyinahandfulofcasesatagenomic79

scale[18].80

Hybridization-capturemethodshavebeenacknowledgedasapromisingwaytoaddress81

boththeallelerepresentationandDNAqualitylimitations[19,20].Suchapproacheshowever82

usuallyrelyonpriorgenome/transcriptomeknowledgeanduntilrecentlyhavebeenlargely83

confinedtomodelorganisms.Addressingthislimitation,therecentdevelopmentof84

UltraConservedElements(UCE[3,5])oranchoredhybridenrichment[21]capture-based85

methodsallowedtargetinghomologouslociatbroadphylogeneticscalesusingonesetof86

probes.Ithoweverrequiresatime-consumingdesignandcostlysynthesisoftheprobesfor87

capturingtheDNAsequencesofinterest.Similarly,exoncapturetechniques,recentlyappliedin88

thefieldofmuseumgenomics[18],requirefreshspecimensforRNAextractionorsynthesizing89

theprobesbasedontheknowntranscriptome.90



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Here,wepresentanapproachwecalled‘hybridizationRAD’(hyRAD),inwhichDNA91

fragments,generatedusingdoubledigestionRADprotocol(ddRAD[8])appliedtofresh92

samples,areusedashybridization-captureprobestoenrichshotgunlibrariesinthefragments93

ofinterest.OurmethodthuscombinesthesimplicityandrelativelylowcostofdevelopingRAD-94

sequencinglibrarieswiththepowerandaccuracyofhybridization-capturemethods.This95

enablestheeffectiveuseoflowqualityDNAandlimitstheproblemscausedbysequence96

polymorphismsattherestrictionsite.Moreover,utilizingstandardddRADandshotgun97

sequencingprotocolsallowsapplicationofthehyRADprotocolinlaboratoriesalreadyutilizing98

theabovementionedmethods,forlittlecost.99

Inshort,thehyRADapproachconsistsofthefollowingsteps(Fig1):100

1)generationofaddRADlibrarybasedonhigh-qualityDNAsamples,narrowsize101

selectionoftheresultingfragmentsandremovingadaptersequences;102

2)biotinylationoftheresultingfragments,hereaftercalledtheprobes;103

3)constructionofashotgunsequencinglibraryfromDNAsamples(eitherfreshor104

degradedasinmuseumspecimens);105

4)hybridizationcaptureoftheresultingshotgunlibrariesontheprobes;106

5)sequencingofenrichedshotgunlibrariesandoptionallyoftheddRADlibrary107

(probesprecursor)forfurtheruseasareference;108

6)bioinformatictreatment(Fig2):assemblyofthereadsintocontigs,alignmenttothe109

sequencedddRADlibraryordenovoassembly,SNPcalling.110



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111

Fig1.Lab-workprocedureusedforhyRADdevelopment.Homologousreadsfrom112

shotgungenomiclibrariesarecapturedthroughhybridizationonrandomRAD-basedprobes.113

Thesefragmentsarethenseparatedusingstreptavidin-coatedbeadsandsequenced.114

I) Hybridization

RAD-seq libraries preparation & size selection

Adaptors removal & biotin labelling

B) RAD-seq probes generation (from high quality DNA)

II) Capture on streptavidin beads

IV) Libraries enrichment

A) Shotgun libraries preparation (DNA to be captured)

DNA of interest

III) Wash

C) Hybridization- capture

Probes sequencing

Final sequencing libraries

Contaminantor undesired loci



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115

Fig2.BioinformaticpipelineusedforprocessinghyRADsequences.First,thereadsare116

demultiplexedandcleaned.Differenttypesofreferenceswerebuiltandthecapturedfragments117

weremappedonthereference.TheSNPsarethencalledaftercorrectingforpost-mortemDNA118

damages.119

120

Inthispaper,wedescribethelaboratoryandbioinformaticpipelinesforobtaining121

hyRADdata,aswellasvalidatetheusefulnessofthemethodontwoempiricaldatasets.Wefirst122

testthemethodontheDNAobtainedfrommuseumandfreshspecimensofLycaenahelle123

butterflies.Weexploredifferentbioinformaticapproachesforassemblinglocioutofthe124

hybridization-capturelibraries,namely:i)mappingcapturedlibrariesonpreviouslysequenced125

RADlocifromfreshsamples;ii)usingRADlociasseedsandthecapturedlibraries’readsto126

extendtheRADlociinordertoobtainlongerlociformapping;iii)denovoassemblyofthe127

Data

Mapcapturesbow.e2

CallSNPsFreeBayes

Cleantrimmoma.c

Clusterprobestolocivsearch

AssemblecapturesfromfreshsampleSOAPdenovo

RescaleBAMwithpostmortaldamagesmapDamage

Demul>plexpairedreadsbybarcodesea-u.lsfastq-multxunzip Qualitycontrol

fastqc

VCFtoNEXUSPGDSpider

VCFtostructureVcf-consensus

reads

Referencecrea.on–differenttypes

reads

Dataprepara.on

SNPsearch

Output

FilterSNPsvcNools

ElongatelociPriceTI



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capturedreadsfromasingle,well-preservedbutterflyspecimenforthereference.Secondly,asa128

proofofconcept,weapplytheprotocoltomuseumandfreshsamplesofOedaleusdecorus,a129

Palearcticgrasshopperspeciesforwhichamarkedeast-westspatialgeneticstructurehasbeen130

identifiedinapreviousstudy[22].131

MaterialsandMethods132

133

Studyspeciesandstudydesign134

Forthefirststepofthemethoddevelopment,weusedsamplesofthebutterflyLycaenahelle135

(Lepidoptera,Lycaenidae)(Table1).Threerecentlycollected,ethanol-preservedsamplesfrom136

Romania,FranceandKazakhstanwereusedforgeneratingtheRADprobes,inordertocover137

variationwithinthefullspeciesrange.Genomiclibrariestobeenrichedbysequencecapture138

werebuiltusingeightsampleswhichincludedsevenmuseumdry-pinnedspecimensfrom139

Finland(4collectedin1985and3collectedin1957)andonerecentlycollectedandethanol-140

preservedspecimenfromRomania.Usingtheseeightsampleswecomparedtheoutputs141

betweenfreshandhistoricalDNAofdifferentage,andtestedtheimportanceofDNAsonication142

ineachcase.ThemuseumsampleswereloanedfromtheFinnishMuseumofNaturalHistoryin143

Helsinki,andtheethanol-preservedsampleswereobtainedfromRogerVila’sButterfly144

DiversityandEvolutionlab(InstituteofEvolutionaryBiology,CSIC,Barcelona,Spain).145

146 147



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Table1.Lycaenahellesamplesusedinthestudy.148

Typeofpreservation

Yearofcollection

DNAconcentration[ng/ul]

Locality

dry(pin-mounted) 1957 2.12 Kuusamo,Finland







ethanol 2007 2.12 DumbravaVadului,Romania 149

Forthemethodvalidation,weused53samplesofthegrasshopperOedaleusdecorus,150

including49samplesofbothfreshandmuseumcollectionspecimensforconstructinggenomic151

librariesforthecapture(seeTable2),andfourfreshspecimensspanningthespecies’152

distribution(Switzerland,Spain,Hungary,Russia)forgeneratingtheRADprobes.Themuseum153

sampleswereonaverage64-years-old,withtheoldestsampledatingbackto1908andwere154

providedbytheNationalHistoryMuseumofLondon(UK),theNaturalHistoryMuseumofBern155

(Switzerland),theZoologicalMuseumofLausanne(Switzerland),theETHEntomological156

Collection(Switzerland),theNaturalHistoryMuseumofBasel(Switzerland),theNatural157

HistoryMuseumofGeneva(Switzerland),andtheNaturalHistoryMuseumofZurich158

(Switzerland).ThefourfreshgrasshoppersampleswereprovidedbyG.Heckel(Universityof159

Bern).160

161

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Table2.SummaryofOedaleusdecorussamplesusedinthestudy.163

Typeofpreservation

Meanyearofcollection(range)

MeanDNAconcentration[ng/ul]±SD(range)

Localities

ethanol 2006(2005-2009) 28.34±28.04(3.2-105.7) Croatia,France,Italy,Russia,Spain,Switzerland

dry(pin-mounted) 1952(1908-1997) 18.31±21.73(0.3-121.4) Algeria,France,Greece,Italy,Madeira,,Portugal(mainlandandMadeira),Spain(mainlandandCanaryislands),Switzerland,Turkey

164

DNAextraction165

DNAwasextractedfrominsectlegsforallthesamples.Asmuseumspecimensare166

usuallycharacterizedbylow-contentofdegradedDNA,theisolationprotocolwasoptimized167

accordingly.ThesampleswereextractedusingQIAampDNAMicrokit(Qiagen,Hombrechtikon,168

Switzerland)inalaboratorydedicatedtolow-DNAcontentsamplesattheUniversityof169

Lausanne,Switzerland.Forthesesamples,DNArecoverywasimprovedbyprolongedsample170

grinding,overnightincubationinthelysisbufferfor14handfinalDNAelutionin20μlofthe171

bufferwithgradualcolumncentrifugation.Extractionoffreshsampleswasperformedusing172

DNeasyBlood&TissueKit(Qiagen).DNAextractionandlibrarypreparationusingmuseum173

specimenswasperformedusingconsumablesdedicatedtothemuseumspecimensonly.174

Bencheswerethoroughlycleanedwithbleachandfiltertipswereusedatallstagesoflabwork.175

RADprobespreparation176

Theprobeprecursorswerepreparedusingadouble-digestionRADprotocol[8,23],177

withfurthermodifications.178

TotalgenomicDNAwasdigestedat37°Cfor3hoursina9μlreaction,containing6μlof179

DNA,1xCutSmartbuffer(NewEnglandBiololabs-NEB,Ipswich,MA,USA),1UMseI(NEB)and180

2UofSbfI-HF(NEB).ThereactionproductswerepurifiedusingAMPureXP(BeckmanCoulter,181

Brea,USA),witharatioof2:1withthesample,accordingtothemanufacturer’sinstructions,182



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andresuspendedin10μlof10mMTrisbuffer.Subsequently,adapterswereligatedtothe183

purifiedrestriction-digestedDNAina20μlreactioncontaining10μloftheinsert,0.5μMof184

RAD-P1adapter,0.5μMofuniversalRAD-P2adapter,1xT4ligasebuffer,and400UofT4DNA185

ligase(NEB).AdaptersequencesareshowninTable3;singlestrandadapteroligonucleotides186

areannealedbeforeusebyheatingto95°Candgradualcooling.Ligationwasperformedat16°C187

for3hours.ThereactionproductswerepurifiedusinganAMPureXPratio1:1withthesample,188

andresuspendedin10mMTrisbuffer.Theligationproductwassize-selectedusingthePippin189

Prepelectrophoresisplatform(SageScience,Beverly,USA)withapeakat270bpand‘tight’size190

selectionrange.191

192Table3.Oligonucleotidesusedintheprotocol.x=barcodesequenceintheadapters;barcode193sequencescanbedesignedusingpublishedscripts[24],availableat:194https://bioinf.eva.mpg.de/multiplex/;I=inosineintheregioncomplementarytothebarcodein195blockingoligonucleotidessequences.196

RADprobesP1adapters,SbfI-compatible(RAD-P1)

RAD-P1.1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTxxxxxxCCTGCA

RAD-P1.2 GGxxxxxxAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT

RADprobesP2adapter,MseI-compatible(RAD-P2)

RAD-P2.1 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

RAD-P2.2 TAAGATCGGAAGAGCGAGAACAA

ShotgunlibraryP1adapters

P1.1 ACACTCTTTCCCTACACGACGCTCTTCCGATCTxxxxxx

P1.2 xxxxxxAGATCGGAAGAGC



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ShotgunlibraryP2oligonucleotide

P2 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCCC

PCRprimers

ILLPCR1 AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTT

ILLPCR2_01 CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_02 CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_03 CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_04 CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_05 CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_06 CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_07 CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_08 CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_09 CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_10 CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_11 CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGC

ILLPCR2_12 CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGC

Blockingoligonucleotides

BO1.P5.F AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

BO2.P5.R AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT

BO3.P7.F CAAGCAGAAGACGGCATACGAGATIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

BO4.P7.R AGATCGGAAGAGCACACGTCTGAACTCCAGTCACIIIIIIATCTCGTATGCCGTCTTCTGCTTG

197

TheresultingtemplatewasamplifiedbyPCRina10μlmixconsistingof1xQ5buffer,198

0.2mMofeachdNTP,0.6μMofeachprimer(Table3),and0.2UQ5hot-startpolymerase199

(NEB).Thethermocyclerprogramincludedinitialdenaturationfor30secat98°C;30PCR200

cyclesof20secat98°C,30secat60°C,and40secat72°C;followedbyafinalextensionfor10201

minat72°C.Inordertoobtainsufficientamountofmaterialfortheprobes,thereactionhadto202

beruninreplicates.Thenecessarynumberofreplicateshastobedeterminedempiricallyto203

reachintotal500-1000ngoftheamplifiedproductrequiredforeachcapture.DNAamounts204



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wereassessedusingaQubitfluorometer(Waltham,MA,USA).Successofsizeselectionandof205

PCRreactionswasconfirmedbyrunningthemonaFragmentAnalyzer(seeS1Fig;Advanced206

Analytical,Ankeny,IA,USA;seeS1Fig).Afterwards,thePCRproductswerepooledandpurified207

usingAMPureXP,witharatioof1:1withtheamplifiedDNAvolume.208

Analiquotoftheresultinglibrarywassequencedandtherestofthelibrarywas209

convertedintoprobesbyremovingadaptersequencesbyenzymaticrestrictionfollowedby210

biotinylation.Theprobeprecursorswereincubatedat37°Cfor3hoursina50μlreaction211

containing30μlofDNA,1xCutSmartbuffer(NEB),5UofMseI(NEB)and10UofSbfI-HF212

(NEB),replicatedasrequiredbytheamountoftheamplifiedproduct.Thereactionwasended213

with20minenzymeinactivationat65°Cfor20minandtheresultingfragmentswerepurified214

usingAMPureXP:reactionvolumeratioof1.5:1.Purifiedfragmentswerebiotinnick-labelled215

usingBioNickDNALabelingSystem(ThermoFisher,Waltham,MA,USA)accordingtothe216

supplier’sinstructionsandpurifiedusingAMPureXP:reactionvolumeratioof1.5:1.The217

resultingfragmentswillthereafterbereferredtoasprobes.218

Shotgunlibrarypreparation219

Shotgunlibrarieswerepreparedfromthefreshandmuseumspecimensbasedona220

publishedprotocolfordegradedDNAsamples[15],modifiedinordertoincorporateadapter221

designofMeyer&Kircher[24].Theapproachusedforlibrarypreparation,utilizingbarcoded222

P1adapterand12indexedP2PCRprimers,allowsahighsamplemultiplexingonasingle223

sequencinglane(seeTable3[24]).224

ForL.helle,DNAfromeachindividualwasdividedintwoaliquots.Onealiquotwaskept225

intact(i.e.highmolecularweightDNAinthefreshsampleandnaturallydegradedDNAin226

museumspecimens)andthesecondwassonicatedusingCovarisfocusedultrasonicator227

(Woburn,MA,USA)withapeakat300bp.Bothaliquotswereprocessedinparallelduring228



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subsequentstepsoflibrariespreparation.AllthelibrariesfromO.decoruswereprepared229

withoutsonication,basedonthetestresultsobtainedfromtheL.hellelibraries.230

DNAsampleswerefirst5’-phosphorylatedinordertoallowadapterligationinthenext231

stepsoftheprotocol.8μlofDNAwasdenaturedat95°Cfor10minutesandquicklychilledon232

ice.The10μlreactionconsistingofdenaturedDNA,1xPNKbufferand10UofT4polynucleotide233

kinase(NEB)wasincubatedat37°Cfor30minandheat-inactivatedat65°Cfor20min.The234

DNAwasthenpurifiedusinganAMPure:reactionvolumeratioof2:1andresuspendedin10μl235

of10mMTrisbuffer.236

Aguanidinetailingreactionofthe3’-terminuswasperformedafterheatdenaturationof237

DNAat95°Cfor10minutesandquicklychillingonice.Thereactioncomposedof1xbuffer4238

(NEB),0.25mMcobaltchloride(NEB),4mMGTP(LifeTechnologies),10UTdT(NEB)and10µl239

ofdenaturedDNAin20µlreactionvolumewasincubatedat37°Cfor30minandheat-240

inactivatedat70°Cfor10min.241

ThesecondDNAstrandwassynthesizedusingKlenowFragment(3’→5’exo-),witha242

primerconsistingoftheIlluminaP2sequenceandapoly-Csequencehomologoustothepoly-G243

tail(seeTable3)addedtotheDNAstrandinthepreviousreaction.A10μlreactionmix244

consistingof1μlofNEBuffer4(10x),0.6μlofdNTPmix(25mMeach),1μloftheP2245

oligonucleotide(15mM),5.4μlofwater,and2μlofKlenowFragment(3’→5’exo-;NEB,5246

U/μl)wasaddedtothe20μloftheTdTreactionmix,incubatedat23°Cfor3h,andheat-247

inactivatedat75°Cfor20min.Thedoublestrandedproductwasthenblunt-endedbyaddinga248

mixconsistingof0.5μlofNEBuffer4(10x),0.35μlofBSA(10mg/ml),0.2μlofT4DNA249

polymerase(NEB,3U/μl)and3.95μlofwater,andincubatedat12°Cfor15min.Theresulting250

productwaspurifiedusingAMPureXP:reactionratioof2:1andresuspendedin10μlof10mM251

Trisbuffer.252

BarcodedP1adapters(seeTable3)wereligatedtothe5’-phosphorylatedendofthe253

double-strandedproductina20μlreactionconsistingof10μlofthedouble-strandedDNA,1μl254



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ofthe25uMadapters,1xT4DNAligasebuffer,and400UofT4DNAligase(NEB).Adapters255

havetobeannealedbeforeuseintheRADprobesprotocol.Thereactionwasincubatedat16°C256

overnight.TheresultingproductwaspurifiedusinganAMPure:reactionratioof1:1and257

resuspendedin20μlof10mMTrisbuffer.LigatedP1adapterswerefilled-inina40μlreaction258

consistingof20μlofpurifiedligationproduct,1xThermoPolreactionbuffer(NEB),12UofBst259

polymerase(NEB),anddNTPs(0.25mMeach),andincubatedat37°Cfor20min.260

TheresultingtemplatewasamplifiedbyPCRadding15μlofamixconsistingof5μlof261

Q5reactionbuffer(5x),0.2μlofdNTPs(25mMeach),2.5μlofthePCRprimermix(5μMeach),262

and0.5UofQ5HotStartHigh-FidelityDNApolymerase(NEB)tothe10μlofthetemplate.The263

programstartedwith20secat98°C,followedby25cyclesof10secat98°C,20secat60°C,and264

25secat72°C,followedbyafinalextensionfor2minat72°C.SuccessofeachPCRreactionwas265

checkedusinggelelectrophoresis,andtheresultingproductswerepurifiedusingAMPure266

XP:reactionratioof0.7:1.Sampleswerethenpooledinequimolarratios.267

Insolutionhybridizationcapture,libraryreamplificationandsequencing268

Thehybridizationcaptureandlibraryenrichmentstepsdescribedbelowarebasedon269

previouslypublishedprotocols[13,25]withsomemodifications.Thehybridizationmix270

consistedof6xSSC,50mMEDTA,1%SDS,2xDenhardt’ssolution,2μMofeachblocking271

oligonucleotide(topreventhybridizationofadaptersequences;seeTable3),500to1000ngof272

theprobesand500to1000ngoftheshotgunlibraries,inatotalvolumeof40μl.Onaccountof273

grasshopperlargergenomesizeandpreliminaryresultsindicatinglowsignal-noiseratiointhe274

butterflylibraries,500ngofhumanCot-1DNA(ThermoFisherScientific,Switzerland)was275

addedtotheO.decorushybridizationmixinordertopreventnon-specifichybridizationscaused276

byrepetitivesequences.Themixwasdenaturedat95°Cfor10minandsubsequentlyincubated277

at65°Cfor48hours.Theprobes,hybridizedwithtargetedfragmentsofthelibrary,werethen278

separatedonstreptavidinbeads(DynabeadsM-280,LifeTechnologies).10μlofthebeads279



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solutionwaswashedthreetimesonthemagnetwith200μlofTENbuffer(10mMTris-HCl7.5,280

1mMEDTA,1MNaCl)andresuspendedin200μlofTEN.40μlofthehybridizationmixwas281

addedtothe200μlofthebeadssolutionandincubatedfor30minatroomtemperature.After282

separatingthebeadswiththemagnet,thesupernatantwasremovedandthebeadswere283

washedfourtimesunderdifferentstringencyconditionsasfollows.Thebeadswere284

resuspendedin200μlof65°C1xSSC/0.1%SDSwashbuffer,incubatedfor15minat65°C,285

separatedonthemagnetandthesupernatantwasremoved.Theabovestepwasperformed286

againwith1xSSC/0.1%SDS,followedby0.5xSSC/0.1%SDSand0.1xSSC/0.1%SDS.Finally,287

thehybridization-enrichedproductwaswashed-offfromtheprobesbyadding30μlof80°C288

waterandincubatingat80°Cfor10min.289

Enrichmentofthecapturedlibrarieswasperformedina50μlPCRreactioncontaining290

1xQ5reactionbuffer(NEB),0.2mMdNTPs,0.5μMofeachPCRprimer(theP1universalprimer291

andoneofthe12P2indexedprimers,seeTable3),1UofQ5HotStartHigh-FidelityDNA292

Polymerase(NEB),and15μlofthetemplate.Theprogramstartedwith20secinitial293

denaturationat98°C;followedby25PCRcyclesof10secat98°C,20secat60°C,and25secat294

72°C;andafinalextensionfor2minat72°C.Theenriched-capturedlibrarieswerepurified295

usinganAMPureXP:reactionratio1:1andpooledinequimolarratiosforsequencing(seeS2Fig296

foraprofileexampleofthere-amplifiedcapturelibraryafterAMPurepurification).297

Theprobesprecursors(RADlibrary)forthebutterflylibrariesweresequencedonone298

laneofIlluminaMiSeq300bpsingle-end.Butterflycapture-enrichedlibrariesweresequenced299

ononelaneofMiSeq150bppaired-end,andgrasshoppercapture-enrichedlibrarieswere300

sequencedononelaneofIlluminaHiSeq100bppaired-end.301

Updatedversionsofthelabprotocolcanbefoundat302

https://github.com/chiasto/hyRAD.303

304



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Dataanalysis305

ThehyRADdatasetscorrespondtotarget-enrichedlibrariesandcannotbeanalysedwiththe306

usualRADpipelines[26,27]Indeed,althoughtheyweregeneratedusingRADloci,theobtained307

sequencesarenotflankedbytherestrictionsitesandinsteadmaynotoverlapcompletely308

and/orextendbeforeandaftertheRADlocus.Asaresult,theanalysispipelinemustincludethe309

followingsteps:310

1)demultiplexingandcleaningofrawreads;311

2)buildingofreferencesequencesforeachRADlocus;312

3)alignmentofreadsagainsttheobtainedreferences;313

4)SNPcalling.314

AllbioinformaticstepsofthehyRADpipelinecanberunathttps://insidedna.me.315

Demultiplexinganddatapreparation316

Theobtainedreadsweredemultiplexedusingthefastx_barcode_splittertoolfromthe317

FASTX-Toolkitpackage[28].RAD-seqsequences(probeprecursors)wereprocessedbyTrim318

Galore![29]andcleanedwiththefastq-mcftoolfromea-utilspackage[30]toremovelow319

qualitynucleotidesandadaptersequences.ThePCRduplicateswereremovedfromRAD-seq320

probesprecursorsandhyRADdatasetsusingtheMarkDuplicatestoolofPicardtoolkit[31].321

ReadsfromhyRADlibrariesweretestedforexogeneousDNAcontaminationusingBLAST322

againstNCBInucleotidedatabase(50,000readsforsonicatedornon-sonicatedfreshor323

museumDNAsamples).324

ExploringthemethodsofreferencecreationonLycaenahellelibraries325

Paired-endreadsobtainedfromthehybridization-capturelibraryforeachsamplewere326

mappedontothreereferences:(1)consensussequencesfortheclusteredRAD-seqreads(RAD-327

ref),(2)RAD-seqreadsextendedusinghybridization-capturedreads(RAD-ref-ext),and(3)328

contigsassembledfromthereadsofhybridization-capturedsamples(assembly-ref).Aswehad329



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noreferencegenomeavailable,wefocusedoncheckingthenumbersofloci/SNPsobtainedby330

mappingthereadsoneachreferenceandtheoverlapofthelociobtainedusingdifferent331

methods.Theoretically,weshouldbeabletoretrieveallthelocifromthesequencedprobe332

precursors(RAD-seqlibrary)inthecapturedlibraries.Wethusevaluatethenumberof333

capturedlocihomologoustothoseretrievedbysequencingtheRAD-seqlibraries.However,334

differentfactorsaffectsignaltonoiseratio(i.e.theproportionoffragmentshomologouswith335

theprobe)inthecapturedlibrariesandcandecreasethenumbersofhomologousfragments336

retrieved.337

VsearchRADlociclustering(RAD-ref).HighqualityreadsofRADprobeswere338

clusteredbysimilarityusingVsearch[32]toobtainlociforfurthermappingthereadsfromthe339

hybridization-capturelibraries.BeforeVsearchrun,weconvertedcleanedfastqfilesintofasta340

formatusingthefasta_to_fastqtoolfromthetheFASTX-Toolkitpackage[28].Toobtainthemost341

reliablecontigsacrosssamples,Vsearchwasrunintwoiterations.Duringthefirstiteration,we342

obtainedconsensusclustersatthewithin-individuallevel(i.e.clusteringoftherawreadsfor343

eachsampleindependently).Duringtheseconditeration,Vsearchwasrunontheconsensus344

clustersobtainedfromthefirstiteration.Theseconditerationallowedustoobtainconsensus345

clustersattheamong-individuallevel.ForbothiterationsweranVsearchwithvariousidentity346

thresholds(0.51,0.61,0.71,0.81,0.83,0.91,0.93,0.96,0.98forthewithin-individualleveland347

0.51,0.61,0.71,0.81,0.85,0.88fortheamong-individuallevel)inordertoidentifyanoptimal348

identitythresholdforclustering,i.e.athresholdthatmaximizesthenumberofclusterswitha349

minimalcoverageof2xand3x,respectively.Inallcases,weusedthecluster_fastoptionfor350

clustering.Theconsensussequencesofeachsecondaryclusterwerethenusedaslocus351

referencesinsubsequentalignmentandSNPcallingsteps.352

VsearchRADlociclusteringandextensionusingcapturedreads(RAD-ref-ext).To353

obtainRAD-ref-ext,weiterativelyextendedcontigsoftheRAD-refusingreadsfromthe354

hybridization-capturelibrary(bypoolingreadscontributedbyalltheanalysedspecimens)355



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usingPriceTI[33]with30cyclesofextensionandaminimumoverlapofasequencematchto356

30.Theobtainedreferencesweretrimmedby60bpateachendinordertoremovesequences357

withputativelylow-qualityends.WeappliedthistoughthresholdforRAD-ref-extonly,as358

probesextensioncanbeperformedonverylow-coveragedata,andwethereforewantedto359

keeptheerrorrate(usuallyhigheronbothsequenceends)attheminimum.360

De-novoassemblyfromcapturedreadsonly(assembly-ref).Assemblywas361

performedonthehybridization-capturedreadsofonegoodqualityethanol-preserved,362

sonicated,sample.Onlysequencesobtainedfromthesinglefreshsamplewereused,as363

stringentcleaningparametersinTrimmomatic[34],usedforthereferenceconstruction,ledtoa364

largeloss(upto80%)ofthereadsfromhistoricalsamples(andsuchdatawasthereforeless365

optimalthanthatfromthefreshspecimenforproducingareference).Moreover,usingone366

individualallowsobtainingamorereliablereference,asanyamong-sampledivergencecan367

resultinbubblesinthecontigsandbiastheassemblybyoversplittingalleles.Asaresultwe368

couldhaveobtainedchimericduplicatedlocipresentedindifferentcontigs.Weused369

SOAPdenovoV2.04toassemblecleanedreadsintocontigs[35].370

MappingandSNPcalling371

ReadsfromthehyRADlibrarywerecleanedbyTrimmomaticwithmilderparameters372

thanreadsusedinthedenovoassembly(keeping60-85%ofthereadsinhistoricalspecimens).373

Readmappingwasperformedusingbowtie2-buildforthereferenceindexingandbowtie2for374

mapping[36],PCRduplicateswereremovedusingtheMarkDuplicatestoolfromthePicard375

toolkit[31]andSNPswerecalledwithFreeBayesusingthedefaultparameters[37,38].To376

evaluatethelevelofDNAdamageinmuseumDNAsamples,weusedmapDamage2.0that377

rescalesbasequalityscoresofputativelypost-mortemdamagedbases[39]inordertominimize378

presenceofpost-mortemconversionsintheresultingSNPs.Datasetsforreplicateswere379

mergedandanalysedforSNPswithFreeBayes.ObtainedVCFfileswerefilteredbythevcffilter380



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toolfromthevcflib[40]andVCFtools[41].Intheresultingsetonlybiallelicsiteswithhigh381

quality(PHRED>30),minorallelecountlargerthan1/6ofall,presentinatleast50%ofthe382

samplesandwithaminimumdepthof6werekept,indelswereremoved.Potentialparalogsand383

multi-copysiteswereremovedbasedonacoverageofastandard-deviationthreetimeshigher384

thanthemean[42].VCFformatfiles[43]wereconvertedtoSNP-basedNEXUSfilesusing385

PGDSpiderconverter[44]andtostructuredatafilesforeveryindividualusingthevcf-386

consensustool(seealsoFig2).Updatedversionsofthebioinformaticpipelinecanbefoundat387

https://github.com/chiasto/hyRAD.388

Geneticstructure389

Inordertocheckwhetherdatawasreflectinggeneticstructure,weapplied390

fastStructure,aStructure-likealgorithmadaptedtolargeSNPgenotypedata[45]tothesixfinal391

datasets(RAD-ref,RAD-ref-extandassembly-ref,bothforsampleswithandwithout392

sonication).Theanalyseswereconductedusingasimplepriorandassumingtwogroups(k=2).393

Overlapbetweenassemblyreferences394

ToevaluatethelevelofoverlapamongthethreeassemblyreferencesforL.helle(RAD-395

ref,RAD-ref-ext,andassembly-ref)weusedtheOrthoMCL[46]pipelinefororthologydetection.396

Mostofthepipelinewasrunwiththedefaultparameters,exceptforBlastallandMCLclustering397

steps.Here,weusedmorestringentparametervalues(e-valueof0.0001andMCLwasrunwith398

aninflationparameterof2.0)inordertoreducechancesofdetectingfalseorthologygroups.As399

aresult,weobtainedclustersofcontigsbeingcontributedbythethreeassemblyreferences.We400

thencountedhowmanyoftheseclusters–presumablycorrespondingtohomologousloci–401

weresharedamongtheavailablereferenceassemblyapproaches.Eventually,torevealthe402

numberofRADlocipresentinthereferences,readsoftherawRADlibraryweremappedon403

RAD-refandassembly-refusingbowtie2[36]andlevelsofmappingwerecompared.404

Proofofconcept:applicationofhyRADtoOedaleusdecorus405



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Theutilityofthemethodwasfurthervalidatedusing49museumandfreshsamplesofa406

Palearcticgrasshopperspeciesforwhichamarkedeast-westspatialgeneticstructurewas407

identifiedpreviously[22].Thecatalogwasbuiltbasedoneightspecimensfromthecaptured408

librarythatshowedthelargestnumberofreadsandspannedthespecies’distributionarea409

(Switzerland,Italy,Spain,Russia),usingthemethodthatyieldedthehighestnumberofcontigs410

andproducedconsistentgeneticstructureinL.helle(assembly-ref,i.e.,denovoreferencebuilt411

usingSOAPdenovo,seeResultsandDiscussionsectionandTable4).Thegeneratedcontigswere412

blastedagainstGenBankdatabasesforbacterial,fungalandtechnicalsequenceswitha413

minimumE-valuethresholdof0.1.Endogeneouscontigsofeachsampleswereassembledto414

generatethefinalreferenceusingGeneiousV9.0.2[47].Filtersweresubsequentlyappliedto415

keeponlyhighqualityandinformativeSNPsasfortheL.hellelibraries. ThefinalVCFmatrix416

wasconvertedintoStructureformatusingPGDSpiderV2.0.9.0[44]andpopulationstructure417

wasinferredusingfastStructure[45]usingasimplepriorandassumingtwogroups(k=2).418

QuantumGISV2.4.0wasusedtopresentthegeographicdistributionofthegeneticclusters[48].419

420

Table4.DataonobtainedreferencesforL.helle(RAD-ref,RAD-ref-ext,assembly-ref)andO.421decorus(assembly-ref).422

Reference Numberofcontigs Largestcontig(bp)

Totallength(bp) N50

RAD-ref(L.helle) 25478 544 5445942 209

RAD-ref-ext(L.helle) 24820 851 2613024 98

assembly-ref(L.helle) 304161 2352 35579979 666

assembly-ref(O.decorus) 408851 13103 119789911 321 423

ResultsandDiscussion424

425

Sequencinganddataquality426



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Lycaenahellelibraries.RAD-seqlibrariessequencingyielded14,188,023and427

hybridization-capturelibraries16,636,502rawreads:8,217,522forsonicatedand8,418,980428

fornon-sonicatedsamples.Additionalsequencingofthereferencelibraryfromtheethanol-429

preservedspecimen(usedfortheRAD-refcreation)yielded4,703,744reads.Theproportionof430

readskeptafterqualityfilteringvariedwithsampleageandpreparationmethod.Forthe431

ethanol-preservedsample,89.8%ofreadsfromsonicatedand89.4%fromnon-sonicated432

samplewereretained.Forthe30yearsoldsamplesthemeanwas74.3%and79.6%,andfor58433

yearsoldsamples70.8%and73.8%forsonicatedandnon-sonicatedsamples,respectively.434

Oedaleusdecoruslibraries.Hybridization-capturelibrariesyieldedatotalof435

69,306,042rawreads.Afterqualityfiltering,80.3%ofreadswerekeptamongallthesamples.436

ComparisonofreferencesobtainedforL.hellelibraries437

Proportionofsingle-hitalignmentsandSNPnumbers.Consensusclusteringofthe438

RAD-basedreferencewithinindividualsproducedthelargestnumberofclusterswith2xand3x439

coveragewithaclusteringidentitythresholdof0.91,comparedtootherthresholdvalues.440

Consensusclusteringamongindividualsproducedthebestresultswithaclusteringidentity441

thresholdof0.71(seeS3Fig).442

Thehighestnumber,lengthandthetotallengthofreferencecontigswereobtained443

usingdenovoassemblywithSOAPdenovo(assembly-ref;Table4).BothRAD-basedassemblies444

producedanorderofmagnitudelowernumberofcontigs.Theextensionperformedonthe445

obtainedRADreferencefollowedbytrimmingofadaptersequencesresultedinreferenceswith446

anaverageshorterlength(lowerN50)thanthestartingcontigs—whereaspriceTIextendeda447

largenumberofprobes,thisdidnotreflectinasubstantiallyhigheraveragelocilengthbecause448

offurthertrimmingofobtainedcontigs.449

Thehighestlevelsofsingle-hitalignmentsformostofthesamples,excepttheoldest450

ones,forbothpreparationmethods(sonicatedandnotsonicated)wereobtainedwhenmapped451



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onthesequencedRADlociextendedusingPriceTI(RAD-ref-ext).Thismethodwasfollowedby452

denovoassemblyusingreadsfromthehybridization-capturelibraryfromasinglefresh453

specimen(assembly-ref)andmappingontheRADloci(RAD-ref);althoughthedifference454

betweenthelasttwoapproacheswasnotlarge(Fig3).Onlyfortheoldestsamplesaswellasin455

thenon-sonicatedfreshsample,denovoreferenceprovidedslightlybetterresults.456

457

Fig3.Percentageofthecapturedreadsshowinguniquemappingeventsfordifferent458

typesofDNApreparationsandbioinformaticpipelines.459

460

IntermsofthenumberofSNPsretainedaftercoverage,paralogsandamong-samples461

overlapfiltering,theRAD-refpipelinedetectedthehighestnumbersofloci,regardlessofsample462

ageandpreparation(Fig4).Noclearcorrelationwithsampleagecouldbeobserved,although463

allthemethodsprovidedthelowestSNPnumbersinthefreshsamples–mostlikelyaneffectof464

0.0

2.0

4.0

6.0

8.0

10.0

12.0

fresh museum,30y.o.

museum,58y.o.

fresh museum,30y.o.

museum,58y.o.

Percen

tageofsingle-mappingevents

Sonicatedlibraries Non-sonicatedlibraries

TypeofthesampleandlibrarypreparaEonprotocol

RAD-ref

RAD-ref-ext

assembly-ref



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smallgeneticdistancebetweenthereferencesampleandthefreshspecimens(asthefresh465

samplewasusedbothforthereferenceandthealignedsample,onlyheterozygotesitesaccount466

forSNPshere).AhighernumberofSNPswasdetectedinthesonicatedlibraryonlyforthefresh467

specimens.468

469

Fig4.MeannumberofSNPspersampleobtainedfordifferenttypesofDNA470

preparationsandbioinformaticpipelines.471

472

RAD-sequencingdatasetsdependonthepresenceoftherestrictionsitesandtherefore473

anypolymorphisminsuchsitesleadstoeithermissinglocioralleles.Asourmethoddoesnot474

dependontherestrictionsitepresence,combinedwiththehighnumberofgatheredSNPs,this475

allowsobtaininglargelyfilleddatamatrices.Matrixfullnesswas>50%inallcases:476

- assembly-ref,non-sonicated:66.1%477

0

500

1000

1500

2000

2500

3000

fresh museum,30y.o.

museum,58y.o.

fresh museum,30y.o.

museum,58y.o.

Num

bero

fSNPs

Sonicatedlibraries Non-sonicatedlibraries

TypeofthesampleandlibrarypreparaAonprotocol

RAD-ref

RAD-ref-ext

assembly-ref



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- assembly-ref,sonicated:63.4%478

- RAD-ref,non-sonicated:52.0%479

- RAD-ref,sonicated:69.1%480

- RAD-ref-ext,non-sonicated:63.4%481

- RAD-ref-ext,sonicated:72.5%482

Lackinganobjectivecriterionforassessingthe‘best’performingmethodforbuilding483

thecatalog,wetestedwhichofthethreereferencescreatedthedatasetproducingtheexpected484

spatialdivisionofgeneticstructurebetweenFinnishandRomanianL.hellesamples.Thespatial485

geneticstructureinferredbyfastStructurerevealedthattheeightsamplesaredividedintotwo486

clustersof,respectively,sevenFinnishvs.oneRomaniansampleonlywhenusingthenon-487

sonicatedlibrarymappedtotheassembly-refcatalog.Thisresultisinagreementwiththe488

hypothesisthatwhenusingsonicatedDNA,weareathighriskofincorporatingcontaminant489

DNAwhichcanblurthesignal(MatthiasMeyer,MaxPlanckInstituteforEvolutionary490

Anthropology,Liepzig,DE;personalcommunication).ThisisaninterestingresultasBLAST491

analysesonthesixcatalogsdidnotretrievedifferencesinthelevelofknowncontaminants(see492

below).493

Locioverlapamongtheassemblymethods.About15.2%ofthereferenceloci494

obtainedviadenovoassemblyweresharedwiththosebasedontheclusteringofRAD-seqreads495

(eitherRAD-reforRAD-ref-ext;Fig5).Incontrast,anappreciablefractionoftheobtained496

referenceloci(59.7%)wereuniquetotheassembly-refapproach.WhenmappingrawRAD497

readsonRAD-ref,26.3%didnotmap,42.4%mappedonceand31.3%mappedmorethanonce.498

ThisshowsthatRAD-refsummarizesca.¾ofthereadsfromtheRADdataset.Incontrast,when499

mappingrawRADreadsonassembly-ref,78,3%ofreadsdidnotmap,21,6%mappedonceand500

0,1%mappedmorethanonce.Thisresultshowsthatassembly-refpossiblycontainsthree501

quartersofalllocithatarenothomologoustotheRADprobes.Suchlowsignaltonoiseratio502

(targetedreadstothenumberoftotalreads)ismostlikelyaresultofbackgroundcarryoverin503



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thehybridizationcapturestep,aphenomenonwhichcanhavemanysources.,Itcouldresult504

from‘daisy-chaining’ofthecapturedfragments[49,50],wherepartiallycomplementaryDNA505

moleculeshybridizewiththeotherfragmentsthatarealreadyhybridizedtotheprobes.Wecan506

howeverdiscardthisexplanationasaprimaryreasonforthebackgroundcarryoveras507

extendingofRADprobesdidnotproducelongercontigs(inRAD-ref-extassemblies).Another508

likelyreasoncouldbecarryoverofrandomDNAfragmentswithrepetitivesequences.The509

extentofsuchprocesscanbesignificantlyreducedbyaddingblockingagentstothe510

hybridizationmix(typicallyCot-1aswasperformedfortheO.decorusdataset[51]orsalmon511

spermDNA),aswellasoptimizinghybridizationtemperatureandwashstringencytoincrease512

captureefficiency.Suchdevelopmentsaredesirablebecausetheyincreasethepercentageof513

readsmatchingthelociofinterestandeventuallyimprovetheoverallsequencingcoverage.514

515

516

Fig5.Numberoflociobtainedusingdifferentbioinformaticapproaches,identifiedusing517

theOrthoMCL[42]pipelinefororthologydetection.518



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519

EffectsofsamplepreparationandageonthenumbersofSNPsobtainedandthe520exogeneousDNAcontent521

Readscanbemappedonareferenceeitheroncewithahighestscore(i.e.,single522

mapping)oronmorethanoneregionofreferencewithclosescores(i.e.,multimapping).The523

reasonsformulti-mappingeventscanbebiological(e.g.,paralogsequences)ortechnical524

(splittingsinglelociintomorereferenceloci),neverthelessthesemappingeventscannotbe525

usedforSNPcallingandofferanotherbenchmarkfortheassemblymethodsused.Differencesin526

thenumberofsinglemappingeventsandinthenumbersofSNPsobtainedwerenotsubstantial527

betweensonicatedandnon-sonicatedsamples,anddependedonthesampleageandthe528

bioinformaticpipelineused(Figs3and4).Weexpectedthatmuseumspecimensshould529

performbetterwithoutsonication,astheDNAwasalreadyvisiblyfragmented,andsonicationof530

museumspecimensmayincreasethelevelsofexogenousDNAcontamination(byfragmenting531

intactfungalorbacterialDNAcontaminatingmuseumsamples;M.Meyer,pers.comm.).In532

termsofmappingevents,whereasthefreshsampleusuallyshowedabetterratioofsingle-to533

multi-mappingeventswhensonicated,therewasatrendtowardsahigherpercentageofunique534

mappingeventsfornon-sonicated30-yearsoldmuseumspecimens,onaverage1.3times535

higher,irrespectiveofthereferenceused(thedifferencewaslessclearfor58-yearsoldmuseum536

specimens,anddependedonthereferenceused).Wewouldthereforeadvisenottosonicate537

DNAobtainedfromthemuseumspecimens,whichsignificantlycutsdownthepriceandtime538

requiredforlibrarypreparation,exceptincaseswhennosignsofdegradationareobservable539

ontheDNAprofile.AslevelsofDNAdegradationofcontemporarysamplesmayvary,onemay540

considerthatthesonicationstepshouldbeadvisablewhenworkingwithwell-preservedDNA.541

However,thisisstillanopenquestion,aswhereasBLASTsearchesdidnotretrievehigher542

fractionsofcontaminantsinsonicatedvs.non-sonicatedlibraries,theexpectedpopulation543

structurewasretrievedwasthenon-sonicatedonemappedonassembly-ref.544



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Asoneofthemaintypesofpost-mortemDNAdegradationisdeaminationofcytosines,545

highlydamagedancientormuseumDNAsamplesareusuallycharacterizedbyhigheruracil546

content[52-54].Inclassicallibrarypreparationprotocols,theusageofaproofreading547

polymerasesshouldstallthechainelongationinthepresenceofuracilandthusreducethe548

misincorporationerrorsinthefinaldataset.Ontheotherhandthisapproachmightnotbe549

optimalforhighlydegradedancientDNAsamples,wherealargefractionofDNAfragmentsmay550

carrycytosinetouracilmisincorporations[53].Moreovertheusageofaproofreading551

polymerasedoesnotpreventthemisincorporationscausedbydirectdeaminationof552

methylatedcytosinetothymine,orlesscommondeaminationofguanidinetoadenine[52,54].553

Intheprotocolusedabove[15],thesecondstrandsynthesiswasperformedusingKlenow554

Fragment(3’→5’exo-),lackingproofreadingability,andthusapproximatelyhalfofthe555

resultingDNAfragmentsshouldhavecytosinetouracilmisincorporationsubstitutedfor556

thymine,amplifiablebyproofreadingDNApolymerase.Wethusoptedforabioinformaticpost-557

processingwayoffiltering-outsuchbases.Post-mortemdamageinthesequencedsampleswas558

assessedbymapDamage2.0,whichrescalessequencefilesbydownscalingqualityscoresof559

likelypost-mortemdamagedbases.AssomeSNPsbecamefilteredbylowerqualityscoresafter560

therescaling,thenumberofSNPsisdecreasedaftermapDamage2.0.Weexpectedhigher561

numberofdiscardedSNPsintheoldestsamples,becauseofahigherproportionofDNAdamage562

occurringwithtime.TheproportionofSNPsdiscardedafterapplyingmapDamage2.0wasthe563

highestamongthe58yearsoldsamples(1.46%forRAD-ref;1.89%forRAD-ref-ext;2.36%for564

assembly-ref),althoughrelativelylow,giventhesamples’ageandpreservationtype(seeS1565

Table).566

Itisworthmentioning,thatthehighernumberofSNPsdetectedinlibrariesfrom567

museumspecimens,comparingtothefreshsamples,isnotaneffectofpost-mortemdamage568

(anoppositetrendwasdetectedwiththehigestproportionoftypeIItransitionstotransitions569

[55]andtransversionspresentinthefreshspecimens;S4Fig)570



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Application:spatialgeneticstructureofOedaleusdecorus571

Thedenovoreferencecatalogwascomposedof408,851referencecontigs.TheN50572

lengthwas321bpandthetotallengthwas119,789,911bp.Amongthetotalnumberofcontigs,573

9%wereshowntobeofexogeneousoriginbytheBLASTsearch,eitheragainstfungiand574

bacteriaGenBankdatabasesoragainsttechnicalsequences.Suchalevelofcontaminantsis575

expectedhere,asincontrasttotheL.hellereferences,whichwerebuiltfromfreshsamples,the576

O.decorusassembly-refwasbasedoneightspecimensfromthecapturedlibrary—eitherfresh577

orpin-mounted—thatshowedthelargestnumberofreads.Atotalof4,783,774informative578

siteswereretrievedafterSNPcalling.Afterremovingindelsandlowqualitysites,125,890sites579

wereconserved.Keepingonlybialleliclociwithaminorallelecountofatleast6,withdata580

fullnesshigherorequalto50%ofthesamples,weobtained6,046loci.Finally,weconserved581

2,979SNPsaftertheremovalofpotentialparalogoussites.ThemediandepthforeachSNPwas582

10.Onaverage,eachofthe49sampleswerecharacterizedby1864SNPsandeachSNPwas583

foundin32individuals(62.7%ofmatrixfullness).584

ThespatialgeneticstructureinferredbyfastStructurerevealedtwogeographically585

distinctcladesinthewestandtheeastofthePalearctic(Fig6).Thisresultsupportstheeastern-586

westernsplitpreviouslyhighlightedinOedaleusdecorusbasedonmtDNAamplicons[22].This587

demonstratesthathyRADisareliabletechniquetoinferspatialgeneticstructurefromboth588

freshsamplesandmuseumsamplescollectedatvarioustimepointsinthepast.589



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590

Fig6.SpatialgeneticstructureofO.decorusinferredusingfastStructurewithk=2and591

simplepriors.Coloursdenotethetwodifferentgeneticgroupssupportedbyapreviousstudy592

relyingonmtDNAmarkers[22].593

594

Conclusions595

596Here,wepresentamethodforobtaininglargesetsofhomologouslocifrommuseum597

specimens,withoutanyapriorigenomeinformation.Despitethedifferencesinsingle-mapping598

eventsamongsamplesofdifferentageswereretrieved,theobtainednumbersofSNPswerenot599

significantlydifferentforthetwoageclassesoftheL.hellemuseumspecimens.Weobtained600

aroundathousandofSNPsfromL.hellesamplesupto58yearsold,confirmingthatitcanbe601

successfullyappliedinthefieldofmuseumgenomics.Theapplicationofthecatalog-building602

methodthatwasthemostpromisingforresolvingpopulationgeneticstructure(i.e.,non-603

sonicatedlibrarymappedtoassembly-ref)tothegrasshopperO.decorus,confirmedthe604

usefulnessofhyRADtoretrievephylogeographicdatausingmuseumsamplesuptoone605

hundredyearsold.Ourmethoddoesnotrequiretime-consumingandcostlyprobesdesignand606

synthesis,noraccesstofreshsamplesforRNAextraction,makingitoneofthesimplestand607

moststraightforwardtechniqueforobtainingorthologouslocifromdegradedmuseumsamples.608



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Intheprotocol,weappliedamodifiedshotgunlibrarypreparationmethod,optimized609

fordegradedDNAfrommuseumspecimens[15].However,thecaptureprotocolpresentedhere610

canbeappliedtoanytypeoflibrarypreparation,includingcommercialones,simplifyingthe611

workflowandcuttingdownthepreparationtime.612

Wealsoexploredseveralbioinformaticapproachesforlociassemblyfromthecaptured613

libraries,acrucialstepwhenworkingonorganismswithoutareferencegenome.Identifyingthe614

mostappropriatecatalog-buildingmethodmaydependonthegoalsofeachstudy.Inourcase,615

thepipelinethatwasthebestatidentifyingpopulationstructureinthebutterflywasrelyingon616

anon-sonicatedlibrarymappedtothedenovoreferenceassemblyfromcapturedreadsfroma617

singleethanol-preservedspecimen,usingSOAPdenovoassembler(assembly-ref).Despitethe618

factthatamaximumof26%oftheobtainedsequencesmappedtothereferencesandthe619

proportionofsinglemappingeventswerenothigherthan10%onaverage(Fig3),wecould620

successfullycallaroundathousandoflociineachcase(Fig4),withhighcoverageacrossthe621

samples.622

Importantlyfromthewetlabprotocolperspective,inthehybridizationstep,wehave623

usedblockingoligonucleotidestoprevent‘daisy-chaining’ofcapturedsequencesbyadapter624

sequences’homology.Usingablockingagentpreventingsimilarchainingcausedbyrepetitive625

sequences(Cot-1DNA,appliedtothegrasshopperlibraries,seebelow[51])aswellas626

optimizingtheconditionsofhybridizationandcapturereactionsforincreasedstringency(e.g.,627

bydecreasinghybridizationtemperatureandthestringencyofthewashesusinghigher628

concentrationofSDSand/orlowerconcentrationofSSC)mayfurtherincreasethe629

hybridizationefficiencyandthusthenumbersofreadsmappingonthereferenceandreduce630

thenumberoflow-coverageloci.631

Thedenovoassemblybuildingpipelineproducedthelargestcontigof2,352bpforthe632

butterflyand13,103bpforthegrasshopperdataset.Althoughthemeanlengthoftheassembled633

contigswasmuchsmaller,ourmethodalsoallowsretrievinglongersequencesthanthelength634



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32

oftheprobesused.ThereasonforthisisthatcapturedsequenceshybridizewithotherDNA635

fragmentswithhomologoussequences,flankingtheprobesequence(i.e.,‘daisy-chaining’[49,636

50]).Thismayleadtoenrichmentacrosslargerfractionsofgenome,asideeffectofourmethod,637

thatcanbeutilizedforassemblyoflargercontigsbyusinglongerprobesandcapturinglonger638

targets.639

Themethodpresentedhere,althoughbasedontherestrictionenzymedigestionofDNA640

tocreatetherandomgenomicprobes,doesnotdependontherestrictionsitepresenceinthe641

capturedlibrary.ThisrepresentsasignificantimprovementoverclassicalRAD-sequencing642

datasets,inwhichincreaseinthephylogeneticdistanceamongsamplesiscorrelatedwithan643

increaseinthenumberofmissingsites[56-61],sometimesleadingtoconflictingsignals644

betweenRAD-andcapture-baseddatasets[62],orarecharacterizedbythepresenceofnull645

allelesthatleadtoheterozygosityorFSTunderestimation[9,10].Inthisaspect,ourapproachis646

similartoothercapture-enrichmentprotocols,suchasUltraConservedElements[5]orexome-647

capture[4],withthebenefitofmuchsimplerandlessexpensiveprobegeneration,without648

accesstogenomeinformationorfreshspecimensforRNAisolation.Notrelyingonthepresence649

ofrestrictionsite,themethodpresentedhereshouldbealsousefulforbroaderphylogenetic650

scales,allowingsequencinghomologouslocifrommoredivergenttaxa,whichwouldnotbe651

possibletoretrieveusingclassicalRAD-seqapproaches.652

653

Acknowledgments654

655WethankAlanBrelsford,AliciaMastretta-YanesandPawelRosikiewiczfortheirhelp656

withdevelopingRAD-sequencingprotocols.JairoPatiñotestedearlyversionsoftheprotocol657

andprovidedvaluablefeedback.RogerVilaandGeraldHeckelkindlyprovidedfreshsamplesfor658

thestudy.Wethankthefollowingmuseumcuratorsforprovidingcollectionsamples:Hannes659

Baur(NaturalHistoryMuseum,Bern,Switzerland),AnneFreitag(ZoologicalMuseum,660



https://doi.org/10.1101/025551


33

Lausanne,Switzerland),RodEastwood(ETHEntomologicalCollection,Zurich,Switzerland),661

DanielBurckhardt(NaturalHistoryMuseum,Basel,Switzerland),PeterSchwendinger(Natural662

HistoryMuseum,Geneva,Switzerland),BarabaraOberholzer(NaturalHistoryMuseum,Zurich,663

Switzerland),GeorgeBeccaloni(NaturalHistoryMuseum,London,UK),LauriKaila(Finnish664

MuseumofNaturalHistory,Helsinki,Finland).WealsothankBrentEmersonforhisconstant665

supportduringthedevelopmentofthismethod.666

667 668



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34

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SupportingInformation834

835

S1Fig.ProfileoftheRAD-probesprecursor,theRAD-seqlibrary.Leftpanel,X-axis:836

fragmentsize(semi-logscale);Y-axis:fragmentdensity(RelativeFluorescentUnits).Right837

panel,gel-likerepresentationoftheleftpanel.838

839

S2Fig.Profileofthere-amplifiedcapturelibraryafterAMPurepurification.Leftpanel,840

X-axis:fragmentsize(semi-logscale);Y-axis:fragmentdensity(RelativeFluorescentUnits).841

Rightpanel,gel-likerepresentationoftheleftpanel.842

843

S3Fig.IllustrationoftheclusteringoptimizationofRAD-refassemblyclustering844

thresholdsusingVsearch.X-axis:clusteringthreshold;Y-axis:numberofclusterswith2x(red)845

or3x(greenline)coverage.Thetoppanelshowswithin-sample,whereasthebottompanel846

showsamong-sampleclusteringresults.Theoptimalthresholdoptimizesthenumberofthe847

clusterswith2xand3xcoverage.848

849

S4Fig.ProportionoftypeIItransitionstoallthetransitionsandtransversionsforeach850

ofthereferencecatalog,withandwithoutpost-mortembiascorrection.Thedatashownarefor851

thefreshsamplewithDNAsonicationandthemuseumsampleswithoutsonication.Theleftplot852

showsvalueswithoutandtherightplotwithmapDamage2.0correction.853

854

S1Table.mapDamage2.0resultsbasedoneachofthethreereferencecatalogsforL.855

helleanalyses,withthenumberofobtainedSNPs(withandwithoutapplicationof856

mapDamage2.0).857



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Hybridization Capture Using RAD Probes (hyRAD), a New Tool for … · 2016. 3. 22. · 3 40 Introduction 41 42 With the advent of next-generation sequencing, conducting genomic-scale