New method developments in pollen DNA barcoding for · • DNA barcoding: • The idenEficaon of...
Transcript of New method developments in pollen DNA barcoding for · • DNA barcoding: • The idenEficaon of...
New method developments in pollen DNA barcoding for forensic applica8ons KarenLeanneBell,EmoryUniversityHomelandSecuritySymposiumSeries,UniversityofTexasElPaso,June2016
1
Overview
• Backgroundandaims• TesEngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment
2
Pollen species iden8fica8on
• ApplicaEons:• Forensics• Allergenmonitoring• PollinaEonecology• DeterminingplantcommunitycomposiEon
• Typicalsample:• Mixtureofpollenspecies
• MaybelowquanEty
3
Pollen DNA metabarcoding
• AdvantagesovermicroscopicexaminaEon:• GreatertaxonomicresoluEon
4
SPECIES
FAMILY
GENUS
FAMILY
GENUS
SPECIES
Image:C.Chu
Pollen DNA metabarcoding
• AdvantagesovermicroscopicexaminaEon:• Somespecieslackfeaturesonpollen• Fewpeopletrainedinpalynology
• ManymorecouldbetrainedinpollenDNAmetabarcoding
5
Pollen DNA metabarcoding
• AdvantagesovermicroscopicexaminaEon:• Somespecieslackfeaturesonpollen• Fewpeopletrainedinpalynology• Fasterwithlargenumbersofsamples
6
Pollen DNA metabarcoding
• AdvantagesovermicroscopicexaminaEon:• Somespecieslackfeaturesonpollen• Fewpeopletrainedinpalynology• Fasterwithlargenumbersofsamples
• Disadvantages:• NotquanEtaEve• Speciesmissingfromdatabases• NostandardbioinformaEcspipeline
7
Overview
• Backgroundandaims• Tes$ngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment
8
ITSmatK
PollenDNAmetabarcodingTesEngthemethodwithknownpollenmixtures
9
Pollen DNA metabarcoding: Concepts and defini8ons • DNAbarcoding:• TheidenEficaEonofspeciesbasedonDNAsequenceofastandardgeneregion
• DNAmetabarcoding:• SimultaneousidenEficaEonofallspeciesinamixtureusinghigh-throughputsequencingofastandardgeneregion
10
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
12
Tes8ng the basic pollen DNA metabarcoding method • Howmanyspeciescanbedetectedwithinasample?• Towhatextentisthisaffectedbytaxonomicrelatedness?• Howrarecanaspeciesbeinasamplebeforeitbecomesundetectable?
13
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
14
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
15
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
18
DNA metabarcdoing: ongoing research • BioinformaEcspipelineformixed-ampliconDNAmetabarcodingincludingrbcL• Effectofsequencingdepth• Standardmethodology
19
Overview
• Backgroundandaims• TesEngtheDNAmetabarcodingmethod• Quan$fica$on• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment
20
Quan8ta8ve pollen DNA metabarcoding: goals
21
SpeciesA58%
SpeciesB23%
SpeciesC10%
SpeciesD9%
hnp://remf.dartmouth.edu/imagesindex.html
Quan8ta8ve pollen DNA metabarcoding: benefits • BenefitsforvariousapplicaEons• PollinaEonbiology:• ProporEonofEmeonaparEcularplantspeciesimpactspollinaEoneffecEveness
• Forensics:• Fine-scalegeographic/temporalsignature• MaynotdifferqualitaEvely
22
Quan8fica8on & biases • Sourcesofbias:• PollenpreservaEonbias• DNAisolaEonbias• AmplificaEonbias• Copynumberbias
• PhylogeneEctrends• CorrecEon
23
Quan8fica8on: tes8ng for biases
• PollenpreservaEonbias:• QuanEfyDNAobtainedfromstoredspecimens
• DNAisolaEonbias:• QuanEfyDNAobtainedfromfreshspecimens
• AmplificaEonbias:• Contextdependent• TesEngonmixturesofknownspeciescomposiEon
• Copynumberbias:• rbcLandITS2• qPCRmethods
24
Quan8fica8on: copy number bias
• qPCR:• FollowsPCRamplificaEoninrealEmeusingfluorescentdye
• QuanEfiesnumberofcopiesofgeneofinterest
• Determinecopynumberofchloroplast,ribosomeandsingle-copygene• LookforphylogeneEcpanernsincopynumber
25
Copy number bias: progress
• Primersdevelopedforchloroplastforallmajorplantlineages• PrimersdevelopedforribosomalDNAforallmajorplantlineages• Primersdevelopedforsingle-copynucleargenefor:• Pinustaeda–gymnosperm• Magnoliagrandiflora–basalangiosperm• Hibiscussyriacus–eudicot
26
Copy number bias: qPCR analysis
• Step1:For3specieswithsingle-copynuclearDNAprimers:• UseqPCRtofindcopynumberofchloroplast,ribosome,andsingle-copynucleargene
• Calculatecopynumberperpollengrainforchloroplast&ribosomeusing:• qPCRcopynumberof3genes• Ploidy• Numberofcellsperpollengrain
27
Copy number bias: qPCR analysis
• Step1:For3specieswithsingle-copynuclearDNAprimers:• UseqPCRtofindcopynumberofchloroplast,ribosome,andsingle-copynucleargene
• Step2:Forallotherspecies:• MeasureDNAconcentraEon• UseqPCRtofindcopynumberofchloroplast&ribosome• Calculatecopynumberperpollengrainforchloroplast&ribosomeusing:• qPCRcopynumberof3genes• DNAconcentraEon• Genomesize• Numberofcellsperpollengrain
28
Copy number bias: qPCR analysis
• Step1:For3specieswithsingle-copynuclearDNAprimers:• UseqPCRtofindcopynumberofchloroplast,ribosome,andsingle-copynucleargene
• Step2:Forallotherspecies:• MeasureDNAconcentraEon• UseqPCRtofindcopynumberofchloroplast&ribosome
• For3specieswithsingle-copynuclearDNAprimerscomparecopynumbercalculaEonfrombothmethodsforaccuracy
29
Copy number bias: sampling & phylogene8c analysis
• 100speciesforanalysis:• 25orders,3specieseachindifferentfamilies• 5orders,2species-pairsinsamegenus,onespeciesfromanotherfamily
• TestforphylogeneEcpanernsincopynumber• MakegeneralizaEonsforlineage,order,orfamily
30
Quan8fica8on: DNA isola8on bias
0
10
20
30
40
50
60
70
80
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
DNAyield(ng/uL)
HaploidGenomeSize(pg)
31
Quan8fica8on: other biases
• PollenpreservaEonbias:• Notexamined• Couldbeexaminedusingstoredsamples
• AmplificaEonbias:• Couldbeexaminedwithresultsofknownmixtures• ResultswillonlyapplyundersamePCRcondiEons• Varieswithotherspeciesinmixture
32
Quan8fica8on: ongoing research
• TestrecentlydesignedqPCRprimers• QuanEfycopynumberof100speciesofpollen• DeterminephylogeneEctrendsforallbiases• ApplycorrecEonfactorstoconvertfromproporEonofDNAsequencingreadstoproporEonofpollengrains• IncludecorrecEonfactorsinbioinformaEcspipelines
33
Overview
• Backgroundandaims• TesEngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment
34
Whole Genome Shotgun (WGS) pollen DNA metabarcoding
Random
DNA
RandomDNA
35
WGS pollen DNA metabarcoding
• AdvantagesoverPCR-basedapproaches:• NoamplificaEonbias• Nocopynumberbias• FindsvariaEonbetweenspeciesidenEcalin“barcode”genes
• Currentchallenges:• Genomesizebias• Fewspecieswithgenomedataforreferencelibrary
36
Tes8ng the WGS method
• Howmanyspeciescanbedetectedwithinasample?• Towhatextentisthisaffectedbytaxonomicrelatedness?• Howrarecanaspeciesbeinasamplebeforeitbecomesundetectable?• Howdoesitcomparetomixed-ampliconsequencing?
37
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
39
WGS results: Known pollen mixtures • Resultsbenerthansimulateddata• FewerfalseposiEves• AllspeciesidenEfiedexceptCaryaillinoinensis
40
WGS results: Overall success
• AllspeciescouldbeidenEfiedgivenpresenceinreferencedatabase• Advantagesovermixed-ampliconsequencing:• SpecieslevelidenEficaEonsofspecieswithidenEcalITS2sequences• Artemisiatridentata• Populustremuloides
• DetecEonofspeciesthatamplifiedpoorlywithITS2• Zeamays
• Disadvantagesovermixed-ampliconsequencing:• Speciesabsentfromdatabase:
• Caryaillinoinensis
41
Tes8ng the WGS DNA metabarcoding method: conclusions
• Demonstratedproofofconcept• Successvariedonlywithdatabasepresence• Notaffectedby:
• Speciesrichness• Taxonomicrelatedness
• Rarity• ProbablynotquanEtaEve
42
WGS: ongoing research
• IsitquanEtaEve?• QuanEtaEvecomparisontostandardDNAmetabarcoding• Effectofsequencingdepth• Standardmethodology
43
Overview
• Backgroundandaims• TesEngtheDNAmetabarcodingmethod• QuanEficaEon• Wholegenomeshotgunapproach• Removalofnon-pollenmaterial• Ongoingmethoddevelopment
44
Ongoing method development
• ImprovingbioinformaEcspipelineformixed-ampliconDNAmetabarcoding:• IncludingmulEplegeneregions• CorrecEngforbiases
• Improvingmethodstoremovenon-pollenplantmaterial• DevelopingstandardmethodsforDNAmetabarcodingandWholeGenomeShotgunmethods
45
Possible future direc8ons
• Developingageographicaldatabasetoassistwithdetermininggeographicoriginofpollensamples• Trialingstandardmethodsonknownmixturesthatresembleforensicsamples• Developingstandardmethodsforspecifictypesofforensicandotheranalyses
46
Acknowledgements
47
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