New method developments in pollen DNA barcoding for forensic applica8ons KarenLeanneBell,EmoryUniversityHomelandSecuritySymposiumSeries,UniversityofTexasElPaso,June2016

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Overview

• Backgroundandaims•  TesEngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment

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Pollen species iden8fica8on

• ApplicaEons:•  Forensics•  Allergenmonitoring•  PollinaEonecology•  DeterminingplantcommunitycomposiEon

•  Typicalsample:•  Mixtureofpollenspecies

•  MaybelowquanEty

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Pollen DNA metabarcoding

• AdvantagesovermicroscopicexaminaEon:•  GreatertaxonomicresoluEon

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SPECIES

FAMILY

GENUS

FAMILY

GENUS

SPECIES

Image:C.Chu

Pollen DNA metabarcoding

• AdvantagesovermicroscopicexaminaEon:•  Somespecieslackfeaturesonpollen•  Fewpeopletrainedinpalynology

•  ManymorecouldbetrainedinpollenDNAmetabarcoding

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Pollen DNA metabarcoding

• AdvantagesovermicroscopicexaminaEon:•  Somespecieslackfeaturesonpollen•  Fewpeopletrainedinpalynology•  Fasterwithlargenumbersofsamples

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Pollen DNA metabarcoding

• AdvantagesovermicroscopicexaminaEon:•  Somespecieslackfeaturesonpollen•  Fewpeopletrainedinpalynology•  Fasterwithlargenumbersofsamples

• Disadvantages:•  NotquanEtaEve•  Speciesmissingfromdatabases•  NostandardbioinformaEcspipeline

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Overview

• Backgroundandaims•  Tes$ngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment

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ITSmatK

PollenDNAmetabarcodingTesEngthemethodwithknownpollenmixtures

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Pollen DNA metabarcoding: Concepts and defini8ons • DNAbarcoding:•  TheidenEficaEonofspeciesbasedonDNAsequenceofastandardgeneregion

• DNAmetabarcoding:•  SimultaneousidenEficaEonofallspeciesinamixtureusinghigh-throughputsequencingofastandardgeneregion

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Mixed-speciespollensamplesfromobjectsofinterest Pollenexinelysis

DNAisola/on

Amplifica/onofbarcodingmarker

Species1Species2

SampleASpecies3Species4

SampleB SampleC…

Applyuniqueindextoeachsample

IndexA IndexB IndexC

Poolandsequence

Sortandremoveindexsequences

SampleA:-  Species1-  Species2

SampleB:-  Species1-  Species3-  Species4

SampleC…-  Species1-  Species2-  Species3

DNAsequencedata

SampleA:-  DNAbarcode1-  DNAbarcode2

SampleB:-  DNAbarcode1-  DNAbarcode3-  DNAbarcode4

SampleC…-  DNAbarcode1-  DNAbarcode2-  DNAbarcode3

SearchDNAsequencedatabase

Bell,K.L.,Burgess,K.S.,Okamoto,K.C.,Aranda,R.,andBrosi,B.J.(2016).ForensicScienceInterna/onal:Gene/cs,21:110-116.11

Tes8ng the DNA metabarcoding method • Whytestthemethod?•  Don’tknowhowaccuratethemethodis•  Don’tknowhowmanysamplescanbemulEplexed•  Don’tknowifitisquanEtaEve

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Tes8ng the basic pollen DNA metabarcoding method • Howmanyspeciescanbedetectedwithinasample?•  Towhatextentisthisaffectedbytaxonomicrelatedness?• Howrarecanaspeciesbeinasamplebeforeitbecomesundetectable?

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Tes8ng the method with “knowns” •  Mixturesofvaryingcomplexity:•  Numberofspecies•  Relatedness•  ProporEonofrarestspecies

•  450bpITS2•  500bprbcL•  IlluminaMiSeq:

•  Dual-index96samples/flowcell

•  SpeciesidenEficaEonwithcustomizedbioinformaEcspipeline

Bell,K.L.,Burgess,K.S.,Loeffler,V.M.,Read,T.D.,andBrosi,B.J.(inprep.).TesEngthesuccessofDNAmetabarcodingofpollenforspeciesidenEficaEonandquanEficaEonwitharEficialmixturesofincreasingcomplexity

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ITS2 results: Increasing number of species

2speciesdetected 3speciesidenEfiedtospecies,Zeamaysundetected,2speciesidenEfiedtogenus

Broussone/apapyriferacorrectlyidenEfied,Zeamaysundetected,ArtemisiatridentataidenEfiedtogenus

4speciesidenEfiedtospecies,Zeamaysundetected,2speciesidenEfiedtogenus

2speciesidenEfiedtospecies,Zeamaysundetected,ArtemisiatridentataidenEfiedtogenus

5speciesidenEfiedtospecies,Zeamaysundetected,2speciesidenEfiedtogenus

3speciesidenEfiedtospecies,Zeamaysundetected,ArtemisiatridentataidenEfiedtogenus

6speciesidenEfiedtospecies,Zeamaysundetected,2speciesidenEfiedtogenus

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ITS2 results: Effect of taxonomic relatedness

Samegenus 2speciesdetected

DifferentAPGIIIlineage

2speciesdetected

Samefamily 2speciesdetected

Zeamaysundetected

SameAPGIIIlineage

2speciesdetected

2speciesdetected

Monocotvsdicot 2species

detected16

ITS2 results: Increasing rarity Differentfamilies Samefamily

2speciesdetected 2speciesdetected

2speciesdetected 2speciesdetected

2speciesdetected 2speciesdetected

2speciesdetected 2speciesdetected

2speciesdetected 2speciesdetected

2speciesdetected 2speciesdetected

2speciesdetected 17

Tes8ng the DNA metabarcoding method: conclusions

•  Successvariedacrossspecies•  Successdependentonrarity• Notaffectedby:•  Speciesrichness•  Taxonomicrelatedness

• NotquanEtaEve

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DNA metabarcdoing: ongoing research • BioinformaEcspipelineformixed-ampliconDNAmetabarcodingincludingrbcL•  Effectofsequencingdepth•  Standardmethodology

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Overview

• Backgroundandaims•  TesEngtheDNAmetabarcodingmethod• Quan$fica$on• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment

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Quan8ta8ve pollen DNA metabarcoding: goals

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SpeciesA58%

SpeciesB23%

SpeciesC10%

SpeciesD9%

hnp://remf.dartmouth.edu/imagesindex.html

Quan8ta8ve pollen DNA metabarcoding: benefits • BenefitsforvariousapplicaEons• PollinaEonbiology:•  ProporEonofEmeonaparEcularplantspeciesimpactspollinaEoneffecEveness

•  Forensics:•  Fine-scalegeographic/temporalsignature•  MaynotdifferqualitaEvely

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Quan8fica8on & biases •  Sourcesofbias:•  PollenpreservaEonbias•  DNAisolaEonbias•  AmplificaEonbias•  Copynumberbias

• PhylogeneEctrends• CorrecEon

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Quan8fica8on: tes8ng for biases

• PollenpreservaEonbias:•  QuanEfyDNAobtainedfromstoredspecimens

• DNAisolaEonbias:•  QuanEfyDNAobtainedfromfreshspecimens

• AmplificaEonbias:•  Contextdependent•  TesEngonmixturesofknownspeciescomposiEon

• Copynumberbias:•  rbcLandITS2•  qPCRmethods

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Quan8fica8on: copy number bias

•  qPCR:•  FollowsPCRamplificaEoninrealEmeusingfluorescentdye

•  QuanEfiesnumberofcopiesofgeneofinterest

•  Determinecopynumberofchloroplast,ribosomeandsingle-copygene•  LookforphylogeneEcpanernsincopynumber

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Copy number bias: progress

• Primersdevelopedforchloroplastforallmajorplantlineages• PrimersdevelopedforribosomalDNAforallmajorplantlineages• Primersdevelopedforsingle-copynucleargenefor:•  Pinustaeda–gymnosperm•  Magnoliagrandiflora–basalangiosperm•  Hibiscussyriacus–eudicot

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Copy number bias: qPCR analysis

•  Step1:For3specieswithsingle-copynuclearDNAprimers:•  UseqPCRtofindcopynumberofchloroplast,ribosome,andsingle-copynucleargene

•  Calculatecopynumberperpollengrainforchloroplast&ribosomeusing:•  qPCRcopynumberof3genes•  Ploidy•  Numberofcellsperpollengrain

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Copy number bias: qPCR analysis

•  Step1:For3specieswithsingle-copynuclearDNAprimers:•  UseqPCRtofindcopynumberofchloroplast,ribosome,andsingle-copynucleargene

•  Step2:Forallotherspecies:•  MeasureDNAconcentraEon•  UseqPCRtofindcopynumberofchloroplast&ribosome•  Calculatecopynumberperpollengrainforchloroplast&ribosomeusing:•  qPCRcopynumberof3genes•  DNAconcentraEon•  Genomesize•  Numberofcellsperpollengrain

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Copy number bias: qPCR analysis

•  Step1:For3specieswithsingle-copynuclearDNAprimers:•  UseqPCRtofindcopynumberofchloroplast,ribosome,andsingle-copynucleargene

•  Step2:Forallotherspecies:•  MeasureDNAconcentraEon•  UseqPCRtofindcopynumberofchloroplast&ribosome

•  For3specieswithsingle-copynuclearDNAprimerscomparecopynumbercalculaEonfrombothmethodsforaccuracy

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Copy number bias: sampling & phylogene8c analysis

•  100speciesforanalysis:•  25orders,3specieseachindifferentfamilies•  5orders,2species-pairsinsamegenus,onespeciesfromanotherfamily

•  TestforphylogeneEcpanernsincopynumber• MakegeneralizaEonsforlineage,order,orfamily

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Quan8fica8on: DNA isola8on bias

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70

80

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

DNAyield(ng/uL)

HaploidGenomeSize(pg)

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Quan8fica8on: other biases

• PollenpreservaEonbias:•  Notexamined•  Couldbeexaminedusingstoredsamples

• AmplificaEonbias:•  Couldbeexaminedwithresultsofknownmixtures•  ResultswillonlyapplyundersamePCRcondiEons•  Varieswithotherspeciesinmixture

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Quan8fica8on: ongoing research

•  TestrecentlydesignedqPCRprimers• QuanEfycopynumberof100speciesofpollen• DeterminephylogeneEctrendsforallbiases• ApplycorrecEonfactorstoconvertfromproporEonofDNAsequencingreadstoproporEonofpollengrains•  IncludecorrecEonfactorsinbioinformaEcspipelines

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Overview

• Backgroundandaims•  TesEngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment

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Whole Genome Shotgun (WGS) pollen DNA metabarcoding

Random

DNA

RandomDNA

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WGS pollen DNA metabarcoding

• AdvantagesoverPCR-basedapproaches:•  NoamplificaEonbias•  Nocopynumberbias•  FindsvariaEonbetweenspeciesidenEcalin“barcode”genes

• Currentchallenges:•  Genomesizebias•  Fewspecieswithgenomedataforreferencelibrary

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Tes8ng the WGS method

• Howmanyspeciescanbedetectedwithinasample?•  Towhatextentisthisaffectedbytaxonomicrelatedness?• Howrarecanaspeciesbeinasamplebeforeitbecomesundetectable?• Howdoesitcomparetomixed-ampliconsequencing?

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Tes8ng the method with “knowns” •  Subsetofknownmixturesusedformixed-ampliconsequencing•  Simulateddataforsamemixtures•  IlluminaMiSeq:

•  21indexedsamples/flowcell

•  24-30Mpairedreadsof150bp

•  SpeciesidenEficaEonwithcustomizedbioinformaEcspipeline

Bell,K.L.,Macpherson,M.,Burgess,K.S.,Read,T.D.,andBrosi,B.(inprep.)AssessingthepotenEalforwholegenomeshotgunmethodsinmixed-speciespollenidenEficaEonusingarEficialmixtures 38

WGS results: Simulated data

•  Samemixturesaslaboratorysamples,withexcepEonofthosecontainingCaryaillinoinensis• MixturecomplexiEes:•  Upto8species•  Pairsofvaryingrelatedness•  Downto10%ofpollengrains,1%ofDNAcontent

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WGS results: Known pollen mixtures • Resultsbenerthansimulateddata•  FewerfalseposiEves• AllspeciesidenEfiedexceptCaryaillinoinensis

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WGS results: Overall success

• AllspeciescouldbeidenEfiedgivenpresenceinreferencedatabase• Advantagesovermixed-ampliconsequencing:•  SpecieslevelidenEficaEonsofspecieswithidenEcalITS2sequences•  Artemisiatridentata•  Populustremuloides

•  DetecEonofspeciesthatamplifiedpoorlywithITS2•  Zeamays

• Disadvantagesovermixed-ampliconsequencing:•  Speciesabsentfromdatabase:

•  Caryaillinoinensis

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Tes8ng the WGS DNA metabarcoding method: conclusions

•  Demonstratedproofofconcept•  Successvariedonlywithdatabasepresence•  Notaffectedby:

•  Speciesrichness•  Taxonomicrelatedness

•  Rarity•  ProbablynotquanEtaEve

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WGS: ongoing research

•  IsitquanEtaEve?• QuanEtaEvecomparisontostandardDNAmetabarcoding•  Effectofsequencingdepth•  Standardmethodology

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Overview

• Backgroundandaims•  TesEngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment

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Ongoing method development

•  ImprovingbioinformaEcspipelineformixed-ampliconDNAmetabarcoding:•  IncludingmulEplegeneregions•  CorrecEngforbiases

•  Improvingmethodstoremovenon-pollenplantmaterial• DevelopingstandardmethodsforDNAmetabarcodingandWholeGenomeShotgunmethods

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Possible future direc8ons

• Developingageographicaldatabasetoassistwithdetermininggeographicoriginofpollensamples•  Trialingstandardmethodsonknownmixturesthatresembleforensicsamples• Developingstandardmethodsforspecifictypesofforensicandotheranalyses

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Acknowledgements

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FundingandIns$tu$onalSupport:•  USArmyResearchOffice(grants

W911NF-13-1-0247&W911NF-13-1-0100toBrosi,Read,&Burgess)

•  EmoryUniversity

Individuals:•  BerryBrosi(Emory)•  KevinBurgess(ColumbusState)•  TimRead(Emory)•  CindyChu(Emory)•  EmilyDobbs(Emory)•  JulieFowler(Emory)•  VirginiaLoeffler(Emory)

Donatedplantmaterial:•  AtlantaBotanicalGarden•  MissouriBotanicalGarden•  NewYorkBotanicalGarden•  MontgomeryBotanicalCenter•  RoyalBotanicGardensVictoria

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KarenLeanneBellEmoryUniversitykaren.bell@emory.edu