Post on 06-Jun-2020
Methods Ecol Evol. 2017;1–11. | 1wileyonlinelibrary.com/journal/mee3
Received:11April2017 | Accepted:1June2017DOI: 10.1111/2041-210X.12836
R E S E A R C H A R T I C L E
Long- range PCR allows sequencing of mitochondrial genomes from environmental DNA
Kristy Deiner* | Mark A. Renshaw* | Yiyuan Li | Brett P. Olds | David M. Lodge | Michael E. Pfrender
*Sharefirstauthorship.
DepartmentofBiologicalSciences,EnvironmentalChangeInitiative,UniversityofNotreDame,NotreDame,IN,USA
CorrespondenceKristyDeinerEmail:alpinedna@gmail.comandMark A. RenshawEmail: mrenshaw@hpu.edu
Present addressesKristyDeinerandDavidM.Lodge,DepartmentofEcologyandEvolutionaryBiology,AtkinsonCenterforaSustainableFuture,CornellUniversity,Ithaca,NY,USA
MarkA.RenshawandBrettP.Olds,OceanicInstitute,Hawai’iPacificUniversity,Waimanalo,HI,USA
Funding informationU.S.DepartmentofDefense’sStrategicEnvironmentalResearchandDevelopmentProgram,Grant/AwardNumber:RC-2240
HandlingEditor:DouglasYu
Abstract1. As environmental DNA (eDNA) from macro-organisms is often assumed to behighly degraded, current eDNA assays target small DNA fragments to estimatespeciesrichnessbymetabarcoding.Alimitationofthisapproachistheinherentlackofuniquespecies-specificsingle-nucleotidepolymorphismsavailableforunequivo-calspeciesidentification.
2. Wedesignedanovelprimerpair capableofamplifyingwholemitochondrial ge-nomes and evaluated it in silico for a wide range of ray-finned fishes (Class:Actinopterygii).Wetestedtheprimerpairusinglong-rangePCRandIlluminase-quencing invitroonamock communityof fish species assembled frompoolinggenomic DNA extracted from tissues. In situ we utilized long-range PCR andIllumina sequencing to generate fragments between 16 and 17kb from eDNA extractedfromfilteredwatersamples.Watersamplesweresourcedfromameso-cosmexperimentandfromanaturalstream.
3. Wevalidatedourmethodinsilicofor61ordersofActinopterygii;wesuccessfullysequencedmitogenomesinvitrofromallsixspeciesinourmockcommunity.Insituwerecoveredmitogenomesforallspeciespresentinourmesocosms.Weaddition-ally recoveredmitogenomes from10of12 species caughtat the timeofwatersamplingandtwospeciespreviouslyonlydetectedfromeDNAmetabarcodingofshortDNAfragmentsfromanaturalstream.
4. Successfulamplificationoflargefragments(>16kb)fromeDNAdemonstratesthatnot all eDNA is highly degraded. Sequencingwholemitogenomes from filteredwatersampleswillalleviatemanyproblemsassociatedwithidentificationofspe-ciesfromshort-fragmentPCRamplicon-basedmethods.
K E Y W O R D S
Actinopterygii,eDNA,mitogenomesequencing
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2017TheAuthors.Methods in Ecology and EvolutionpublishedbyJohnWiley&SonsLtdonbehalfofBritishEcologicalSociety
2 | Methods in Ecology and Evolu on DEINER Et al.
1 | INTRODUCTION
TheuseofDNAfoundintheenvironment(eDNA)tocataloguebiodi-versityisgainingmomentum(Creeretal.,2016).Fromsurveyingthethreedomainsoflifeinsoils(Drummondetal.,2015)towhalesintheocean(Footeetal.,2012),biodiversityinformationisbeingproducedonunprecedentedscales.
PerhapsbecausetheuseofeDNAtodetectspecieswaspartiallyinspiredby the fieldof ancientDNA (Thomsen&Willerslev, 2015),researchersassumedthateDNAwashighlydegraded.Becauseofthisassumption,andcoupledwithcurrentsequencelengthlimitationsofnext-generationsequencers(e.g.IlluminaMiSeq),researchershavefo-cusedonproducingsmallfragments(c.50–400basepairsinlength)using a PCR amplicon approach to characterize macro-organismalspecies richness (Olds etal., 2016;Valentini etal., 2016).However,relianceonshort-fragmentPCRampliconsfromeDNAlimitsthecur-rentutilityofthemethodbecausespecies-levelassignmentsareoftennot possible for short reads (Deiner, Fronhofer,Mächler,Walser, &Altermatt,2016;Portetal.,2016).
While itmay be true that some of the eDNA in environmentalsamples is degraded, evidence that not all eDNA is degraded hasemerged.Inarecentstudybasedonwaterfromafishpond,mostoftheeDNAdetectedwasfromparticlesthatrangedinsizefrom1to10 μm,consistentwiththepresenceofintacttissuesorcellsinaquaticenvironments(Turneretal.,2014).ThisresultsuggeststhateDNAforspeciescurrentlyoccupyingahabitat isnotprimarily freeDNAsus-pendedinsolution,butthatitcouldbecellularormembraneboundDNAinatightlycoiledorcircularstateandcomparativelysafefromdegradativeprocesses(Torti,Lever,&Jørgensen,2015).Wethereforehypothesizedthatitshouldbepossibletolong-rangePCRamplifyandsequencewholemacro-organismalmitochondrial genomes (mitoge-nomes)fromDNAisolatedfromwatersamples.
Totestthishypothesis,wedesignedanovelprimersetinthe16Sregion of themitochondrial genome that is nearly 95% conservedat the sequence level across the classofActinopterygii (ray-finnedfishes) and could be used for long-range PCR amplification of fishmitogenomes fromwater samples. Long-range PCR is a viable op-tionfortheenrichmentofwholemitogenomesfromenvironmentalsamplesbecauseinasingleamplificationitcanproduceafragmentthat encompasses the entire mitogenome (Zhang, Cui, & Wong,2012).Theamplificationof theentiremitogenome in a singlePCRhasinherenttimeandcostadvantagesoveramplificationofmultiplefragments, reduces the potential complications of nuclear-encodedmitochondrialpseudogenes(NUMTs)andavoidstherearrangementof targeted priming sites in mitogenomeswith altered gene order(Cameron,2014).However, the successof long-rangePCRamplifi-cationsdependsonthepresenceofrelativelyhigh-qualityandhigh-molecular-weightDNA.
SequencingwholemitogenomesfromeDNAsamplescouldvastlyimprovespeciesassignmentcapabilitiesbecausefull-lengthbarcodes,suchasthecytochromecoxidaseI(COI)regionforanimals(Hebert,Ratnasingham,&deWaard,2003),couldberecoveredandusedforthe identification of species in communities. Other mitochondrial
genes typically used in phylogeography, systematics and conserva-tion genetics could also be recovered in their entirety to provide anon-destructivemethodforsamplingwholecommunitiesforstudies relatedtocommunityphylogeneticsandconservation.
Inthisstudy,wevalidateamethodtoamplifyandsequenceentiremitochondrialfishgenomesfromwatersamples(Figure1).Insilicowetestednewlydesignedmitochondrialprimersandinvitroperformedlong-rangePCRandnext-generationsequencingonresultingamplifi-cationsusingamockcommunityamassedfromtissueextractedDNA.Insituwevalidatedthemethodusingwatersamplescollectedfromamesocosmexperimentwithanassembledfishcommunityofeightspecies, and fromanatural streamknown tohave12speciespres-entatthetimeofsampling.DNAextractionsfromthemesocosmandstreamsampleswerethesameasthoseusedfortwopreviouseDNAstudies of fish communities (Evans etal., 2016; Olds etal., 2016), allowingustocomparethespeciesrichnessestimatedfromashort-fragmentPCRampliconapproachtothatofusinglong-rangePCRandwholemitogenomesequencingfromwatersamples.
2 | MATERIALS AND METHODS
2.1 | Primer design and in silico evaluation
A batch download of Actinopterygii 16S sequences fromGenBankwasalignedusingthePartTreealgorithminMAFFTversion7(Katoh&Standley,2013).ThealignmentwasviewedinBioEdit(Hall,1999)andconservedregionsidentifiedbyeye.Theseregionswerethenevalu-atedinPrimer3(Untergasseretal .,2012)forputativeprimerpairs.PrimersActinopterygii16SLRpcr_F(5′-CAGGACATCCTAATGGTGCAG-3′)andActinopterygii16SLRpcr_R (5′-ATCCAACATCGAGGTCGTAAAC-3′)weredesignedtobeimmediatelyadjacenttooneanothertoPCRam-plifynearlytheentiremitogenomeinasinglereaction.Theonlypartof themitogenomenot amplified is the43-bp area coveredby theActinopterygii16SLRpcrprimingregion.
Toevaluatethepotentialtaxonomiccoverageoftheprimers,theywereconcatenatedintoasinglesequenceinthesamereadingframe(i.e. the reverse primer was reverse-complemented before it wasconcatenated)resultingina43-bpfragment.ThefragmentwasthenalignedwithActinopterygii fishmitogenomes available onMitoFishv3.02 (Figure1a) (Iwasaki etal., 2013). One thousand five hundredandtwelvefishspeciesintheclassActinopterygii,representing62or-dersand310families,wereconsidered(AppendixS1).blastn(blastall2.2.26)wasusedtoalignprimerfragmentstomitogenomeswithout-putintabularformat,e value = 10−4,andwithoutlow-complexityfil-ter(−m8−FF−e1e−4)toensureasinglehitforeachmitogenome(Altschul,Gish,Miller,Myers,&Lipman,1990).Mismatchesbetweeneachprimerand the referencegenomes fromMitoFisharegiven inAppendixS1.
2.2 | In vitro evaluation using mock community
To test the performance of the primers, laboratory methods andbioinformaticpipeline,an invitro testwasperformedusingamock
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community sample at a concentration of 0.6ng/μl (0.1ng/μl from each species) of tissue-derived DNA from six Indo-Pacific ma-rine fishes: Amphiprion ocellaris, Salarias fasciatus, Ecsenius bicolor,Centropyge bispinosa,Pseudanthias dispar and Macropharyngodon ne-grosensis (Figure1b)(Oldsetal.,2016).DNAextractionsforeachofthe sixmock community specieswereperformedwith theDNeasyBlood andTissueKit (Qiagen,Hilden,Germany), following thepro-tocolsasoutlinedbythemanufacturerwiththeexceptionthatfinalelutions were made with 200μl of 1X TE buffer, low EDTA (USBCorporation,Cleveland,OH).Thetissue-derivedDNAwasquantifiedwith theQubit® dsDNABRAssayKit (Life Technologies, Carlsbad,CA),andequalnanogramamountsfromeachofthesixspecieswerecombinedintoasinglemockcommunityDNAextract.
PCRamplificationofthemockcommunitysampleincluded25μl of LongAmp® Taq 2X Master Mix (New England BioLabs, Ipswich,MA),20picomolesofeachprimer(forwardandreverse),5μl of mock communityDNAextract and sterilemolecular gradewater to bringthetotalvolumeto50μl.Cyclingparametersincludedaninitialdena-turationstepat94°Cfor30s;35cyclesofdenaturationat94°Cfor30s,annealingat62°Cfor1min,extensionat65°Cfor14minand10s;andafinalextensionstepat65°Cfor10min.TosequencetheamplifiedPCRproductontheIlluminaMiSeq,theentire50μlPCRam-plificationwaselectrophoresedona0.75%agarosegel,afragmentofexpectedsize(16–18kb)forthemitogenomeswasexcisedfromthe
gelwitharazorblade,thePCRproductwaspurifiedwiththeQIAquickGelExtractionKit(Qiagen)andelutedin50μlAEbuffer.Theresult-ingDNAwasquantifiedwith theQubit® dsDNABRAssayKit (LifeTechnologies).
Based on the Qubit reading for the cleaned PCR product, approximately 200ngwas diluted in a total volume of 52.5μl; thePCRproductwas then shearedwith a S220Focused-ultrasonicator(Covaris,Woburn,MA).Preparationof themockcommunitysamplefor sequencing on the Illumina MiSeq followed the manufacturer’ssuggestedprotocolasoutlinedfortheTruSeqNanoLTSamplePrepKit(low-sampleprotocol)fora550-bpinsertsize(Illumina,SanDiego,CA).SequencingwasperformedwiththeMiSeqReagentKitv3(600cycles; Illumina), producing paired end reads eachwith a length of300 bp.
2.3 | In situ evaluation from water samples
EnvironmentalDNAusedfor thisstudywasextractedfromfilteredwatersamples thatwereutilized in twopreviousstudies: twosam-ples(HighDensity,SkewedAbundance,Tank3[HS3];HighDensity,EvenAbundance,Tank3[HE3])fromamesocosmexperiment(Evansetal.,2016)andeightsamples(R1-D,R1-U,R2-D,R2-U,R3-D,R3-U, R4-D, R4-U) from a stream survey (Figure1c) (Olds etal., 2016).Details of the collection, filtration and DNA extraction have been
F IGURE 1 Methodsoverviewforlaboratoryworkflowusedtodesignandtestlong-rangePCR(LR-PCR)forsequencingofwholemitochondrialgenomesfromenvironmentalDNA.Eachbox(a–d)representsthestepsusedinsilico,invitroandinsitutovalidatethemethod.High-throughputsequencingontheIlluminaMiSeqisabbreviatedasHTS
(a)
(b)
(d)
(c)
4 | Methods in Ecology and Evolu on DEINER Et al.
previouslydescribedinEvansetal.(2016)andOldsetal.(2016).DNAextractswere treatedwithZymoOneStep™PCR InhibitorRemoval(Zymo,Irvine,CA)kitspriortoamplificationofmitogenomesvialong-rangePCR.Amplificationsweredoneexactlyasdescribed in the invitro section with the exception that 20μg bovine serum albumin(VWR,Radnor,PA)wasaddedtothePCRreaction.
BasedonQubitreadingsforthecleanedPCR-amplifiedproducts,1ng(inatotalvolumeof5μl)foreachofthemesocosmsampleswaspreparedforsequencingontheIlluminaMiSeqfollowingthemanufac-turer’ssuggestedprotocolasoutlinedfortheNexteraXTDNALibraryPreparationKit.Foreachoftheeightstreamsamples, librarieswerepreparedasdescribed for the invitro teston themockcommunity.Forall10samples(includingmesocosmandstream),sequencingwasperformedwiththeMiSeqReagentKitv3(600cycles;Illumina),pro-ducingpairedendreadseachwithalengthof300bp.Thetwolibrarypreparationmethodswereusedbecausewewereunsuccessfulinpro-ducingqualitydata for libraries generated from the streamsamplesutilizingtheNexteraXTDNALibraryPreparationKitandfoundtheTruSeqNanoLTSamplePrepKitmethodultimatelytobemorereliablefor thepreparationof Illumina libraries fromeDNAamplifiedwholemitogenomes.
2.4 | Bioinformatic filtering, mapping and de novo assembly
Raw reads were quality filtered by removing Illumina sequenc-ing adaptor, low-quality sequences with average quality less thanQ20 in any 10-bp window and short sequences with length lessthan 50-bp using Trimmomatic v0.32 (Bolger, Lohse, & Usadel,2014) with “ILLUMINACLIP: MiSeq.adapter.fas:3:30:6:1:trueSLIDINGWINDOW:10:20:MINLEN:50.”Readswereusedforalign-mentandassemblyonlywhenbothforwardandreversereadspassedqualityfiltering.Afterqualityfiltering,readsweremergedtoavoid-ing counting the sequencing depth twice based on the sameDNAfragment using USEARCH (Edgar, 2010) v8.1.1861_i86linux32fastq_mergepairs command with minimum overlap length=16 bydefaultandmaximumdifferencepercentage=1%(−fastq_maxdiffpct0.01). If pairedend reads couldbemerged,only themerged readswereusedformappinganddenovoassemblyanalyses.Ifpairedendreads could not bemerged, both forward and reverse reads weremapped tomitogenomes andwere used in the de novo assembly.Therefore, bothmerged and unmerged readsweremapped to the6mitogenomes represented in themock community, the 8mitog-enomes from the twomesocosmdensities (Evans etal., 2016) andthe 14 mitogenomes from fish that were previously captured (orknowntooccur in thewatershed)whenwatersamplesweretakenfromJudayCreek(Figure1d)(Oldsetal.,2016).BWAv0.7.15-r1140(Li&Durbin,2009)withamaximumdifferenceintheseed(−k)equalto2andseedlengthequalto32wasusedformapping.Themissingprobabilitywassetundera0.02errorrate (−n) tobeequalto0.06whichisconsistentwitha97%similarityofOTUclusteringusedforthetaxonomicassignmentofampliconsfrompreviousstudies(Evansetal.,2016;Oldsetal.,2016).SAIformatfilesfrombothendsofthe
reads were combined with “bwa sampe” commandwithmaximuminsert size equal to 1,000bp.Merged readswere aligned as singleendedreadswith“bwasamse”command.Readswithmappingquality(MAPQ)<20wereremoved.Onlyuniquealignedreads(i.e.readswith“XT:A:U”insamfile)wereusedforreferencemapping.Additionally,all reads from JudayCreekwere combinedbeforemapping to ref-erence sequences. Samtools v1.2 (Li etal., 2009) command “stats”wasusedtocalculatenumberof readsmappedforeachreference.The mapping ratio was calculated as (number of mapped mergedreads+number of mapped unmerged reads)/(the total number ofmergedandunmergedreads).Singlenucleotidepolymorphismsweredetermined frommapped reads as described inData S1.BEDtoolsv2.25.0 (Quinlan & Hall, 2010) command “genomecov” was usedfor reference coverage and average sequencing depth calculation.ReferencecoveragewasvisualizedusingGeneiousv.9.1.5andscaf-folds fromde novo assemblyweremapped to the reference usingdefaultvaluesinGeneiousv.9.1.5.Tocheckforcrosscontaminationduringsamplehandling,referencemappingwasdonewithallspeciesused in thisstudyforall libraries.Accessionnumbers for referencesequencesarereportedinAppendixS1.
For themesocosm samples (HE3 andHS3, Evans etal., 2016),reads afterTrimmomatic filteringwere used for de novo assemblywiththemetagenomicsassemblerIDBA-UDv1.1.1(Peng,Leung,Yiu,&Chin,2012).Command“fq2fa”wasusedtotransferfastqformattofastaformatandremoveanyreadwithanunknownbasepair“N.”Command“idba_ud–pre_correction–min_support20”wasusedtoassemblemitogenomes.For themockcommunityandJudayCreeksamples,readsafterTrimmomaticfilteringwerenormalizedbasedonkmerfrequency(Crusoeetal.,2015)becausethesequencingdepthfrom these sampleswas too high.A custom Perl scriptwasmadeto remove readswith sequencingdepthhigher than50×basedon17-mer.Normalized readsandPerl scriptused fordenovoassem-blyareprovidedonDryad (http://dx.doi.org/10.5061/dryad.q5gg0).Normalizedreadswereassembledwiththesameparametersasthemesocosm samples with the metagenomics assembler IDBA-UDv1.1.1. Assembled scaffolds were then mapped to the referencegenomeswithblast+ (Camachoetal .,2009)toestimatecoverageforeachgenebasedonthereferencesusinga95–97%sequencesimilaritycutoff.
2.5 | Long- range PCR compared with amplicon sequencing approach
Readsproducedfromindependentlysequencedgenefragments(16S,12SandCytB)inpreviousstudies(Evansetal.,2016;Oldsetal.,2016)weremappedtothesamereferencemitogenomesusedintheinsituevaluationwith the same parameters as those used for long-rangeamplifiedmitogenomes.Onlyreadsthatpassedqualitycontrolweremapped.Mitogenomecoverageoftheampliconsandsequencedepthwere evaluated and qualitatively compared to the values achievedfromlong-rangeamplifiedproductsfortheentiredenovoassembledgene.Differencesintheestimatedspeciesrichnessbetweenthetwomethodswerealsodocumented.
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3 | RESULTS
3.1 | In silico evaluation of primers
FortheinsilicotestoftheActinopterygii16SLRpcrprimerfragment(i.e. encompassingboth the forwardand reverseprimers), onlyonefishorderaveragedgreaterthantwomismatches,whiletheother61ordersaveragedlessthanorequaltotwomismatches(AppendixS2).Thereverseprimerbindingsitewasmoreconservedthantheforwardprimerbindingsite.Basedonanaveragemismatchcriterionoftwoorlessmismatches, theActinopterygii16SLRpcr primer region has thepotentialtolong-rangePCR-amplifyentiremitogenomesforallordersexcept Osteoglossiformes. However, several of the species withinmanyordershadmismatchesof twoormorebasepairs in the firstthreebasepairsofthe3’endthatcouldleadtofailedamplificationoftheirDNA(AppendixS2).PrimerswerenotassessedforsimilaritytoothertaxonomicgroupsoutsideofActinopterygii.
3.2 | In vitro evaluation and de novo assembly of mitogenomes from mock community
For the mock community experiment, 77.6% of reads mapped tooneofthesixspeciesincludedinthemixtureofDNA(AppendixS3).Nearlywholemitogenomeswererecoveredforallspeciesinthemockcommunity using the reference mapping approach (Table1). SNPswerecalledonlyinonespecies(DataS1).
Usingthedenovoassemblyapproach,asinglescaffoldwaspro-ducedthatcoveredthefull-lengthofthereferencegenomeforthreeofthesixmockcommunityspecies.Fortheotherthreespecies,twoscaffoldsforeachspecieswererecoveredsuchthatwhencombinedtheycoveredbetween97.9%and100%oftheirrespectivereferencemitogenomes (Table1). Additionally, the full length of genes com-monlyused in fisheDNAmetabarcodingwere recovered (AppendixS4).Of the reads thatmapped to speciesnot included in themockcommunity, four species thatwere only in themesocosms and notobserved in Juday Creek (Campostoma anomalum, Fundulus notatus,Gambusia holbrooki and Pimephales promelas) had no readsmappedto their reference (AppendixS5). Several speciesobserved inJudayCreekshowedmoderatelevelsofmappingtotheirreference(0.04%ofreads)eventhoughtheywerenotincludedinthemockcommunitylibrary (AppendixS5).However, thedenovoassembly showed thatnoscaffoldsforspeciesfromJudayCreekcouldberecoveredabove1,900bpinlength(AppendixS5).
3.3 | In situ evaluation from water samples
Forthehigh-density,evenabundance(HE3)andskewedabundance(HS3) mesocosm communities, 65.2% and 75.1% of readsmappedtooneoftheeightspeciesincludedinthemesocosm(AppendixS3).Wholemitogenomes for theeight fishwere successfully recovered(99.5–99.9%, Table1); however, one species,P. promelas, coveragewaslowerintheHE3comparedtoHS3treatment(86.2%vs.99.5%).SNPswerecalledinmostspecies(DataS1).
Using the de novo assembly approach on the mesocosm sam-ples, longscaffoldswereassembledformostspeciesandwerenearcompletemitogenomes(e.g.16,524bpforCatostomus commersonii in HS3,Table1).Denovoresultsweremoreconsistentwhenabundancewas even (HE3). Pimephales promelas de novo assembled scaffolds wereshort inbothmesocosmcommunitiesandseveral specieshadmanyscaffoldsthatcouldnotbemergedintoasingleassemblywith-outguidanceofareferencesequence(Table1).GenescommonlyusedineDNAmetabarcodingstudiesoffishweredenovoassembledforallspeciesevenwhenthewholemitogenomecouldnotbeandrangedincoveragefrom89%to100%(AppendixS4).
ForJudayCreek,46.2%ofreadscouldbeuniquelymappedto10of12speciesconfirmedpresentwhenwatersampleswerecollectedandtwoadditionalspeciespreviouslydetectedonlyfromeDNA(Oldsetal., 2016) (Appendix S3).Whole mitogenomes for 10 of the 12speciescaughtwithelectrofishingweresuccessfully recovered frommappingreadstotheir references (94–100%,Table1,Figure2).ThetwospeciesnotrecoveredwereLepomis macrochirus and Salmo trutta. WeadditionallyrecoveredmitogenomesfromtwospeciespreviouslyonlydetectedfromeDNA,butareknowntooccurinthewatershed(Cyprinus carpio and Micropterus salmoides)(Table1).SNPswerecalledforallspecies(DataS1).
Thedenovoassemblyrecoverednearlywholemitogenomescaf-foldsforabouthalfthespecies (Table1).Thedegreeofcoverageofthesescaffoldstotheirreferencesequencesrangedfromasinglescaf-foldrepresentinganearlycompletemitogenometo29smallerscaf-foldscoveringnearlytheentiremitogenome(Table1,Figure2).Genescommonlyused ineDNAmetabarcodingstudiescouldberecoveredfrom the de novo assemblies and the scaffolds overlapping thesegenescoveredfrom88%to100%ofthefullgenelength(AppendixS4).Somesequencereads fromtheJudayCreeksamplemappedtothereferencegenomesoffishthatwereonlyusedinthemockcom-munity and mesocosm experiment and were not present in JudayCreek (AppendixS5). Inmostcasesthesequencedepthanddegreeofcoverageofthesereadswaslow(0.6%–6.5%),withhighercoverage of references from the mock community species (Appendix S5).Noneofthesespecieshaddenovoassembledscaffoldslongerthan2,100bpinlength.
Comparisons between long-range PCR-amplified mitogenomesandtheshort-fragmentPCRampliconsgeneratedfromthesameDNAextractsrevealedsomespecies(S. trutta and L. macrochirus)wholemi-togenomeswere not detected even though their smaller fragmentswere (Appendix S3). Additionally, two species detected previouslyonly by eDNA,M. salmoides and C. carpio (Olds etal., 2016), werealsodetected;mappingrevealedthattheirwholemitogenomescouldbe recovered and that long fragments couldbedenovo assembled(Table1).
4 | DISCUSSION
We demonstrate that it is possible to sequence whole mitoge-nomesoffishfromDNAextractedfromwatersamplesbycoupling
6 | Methods in Ecology and Evolu on DEINER Et al.
TABLE 1 Long-rangePCR(LRP)ampliconreadmappingstatisticsforspeciesinthemockcommunity,twomesocosmcommunitiesandJudayCreek.Referencecoverageforeachspecies’
mitogenomewasevaluatedinoneofthetwoways:eachreadmappedtoareferenceordenovoassembled(seemethods).Nisthenumberofindividualspresentattimeofsampling(Juday
Creek)orusedinexperiment(MockorMesocosm)
Spec
ies
Com
mon
nam
eRe
fere
nce
leng
th (b
p)N
LRP
map
ped
to re
fere
nce
LRP
de n
ovo
asse
mbl
ed
Uni
quel
y m
appe
d re
ads
Refe
renc
e co
vera
ge (%
)A
vera
ge
sequ
ence
dep
thLo
nges
t sc
affo
ld (b
p)Re
fere
nce
cove
rage
(%)
No.
of
scaf
fold
s
Mockcommunity
Amph
iprio
n oc
ella
risCommonclownfish
16,649
14,559
99.4
7116,652
99.6
1
Cent
ropy
ge b
ispin
osa
Twospinedangelfish
16,772
1798,978
100
11,704
13,633
97.9
3
Ecse
nius
bic
olor
Flametailblenny
16,534
1288,587
100
4,265
13,528
98.7
3
Mac
roph
aryn
godo
n ne
gros
ensis
Blackleopardwrasse
16,889
1539,213
100
8,053
16,890
100
1
Pseu
dant
hias
disp
arDisparanthias
16,954
17,174
100
109
16,888
99.6
1
Sala
rias f
asci
atus
Jewelledblenny
16,496
1318,871
100
4,728
13,867
98.2
4
Mesocosmhigh-density/evenabundance(HE3)
Cam
post
oma
anom
alum
Centralstoneroller
16,649
10122,874
99.9
1,748
14,220
99.2
6
Cato
stom
us c
omm
erso
nii
Whitesucker
16,622
108,167
99.6
118
16,524
99.4
1
Cypr
inus
car
pio
Commoncarp
16,575
10123,924
99.9
1,770
14,479
99.4
4
Fund
ulus
not
atus
Blackstripetopminnow
16,510
1053,266
99.9
781
16,149
100
2
Gam
busia
hol
broo
kiEasternmosquitofish
16,611
1075,672
99.9
1,068
10,229
100
6
Lepo
mis
mac
roch
irus
Bluegill
16,489
1016,677
99.9
231
16,509
100
1
Pim
epha
les p
rom
elas
Fatheadminnow
16,709
1022,247
99.5
318
1,618
53.6
36
Sem
otilu
s atr
omac
ulat
usCreekchub
16,623
1032,941
99.9
472
9,282
99.0
5
MesocosmHigh-density/Skewedabundance(HS3)
Cam
post
oma
anom
alum
Centralstoneroller
16,649
4105,169
99.9
1,528
7,956
97.7
23
Cato
stom
us c
omm
erso
nii
Whitesucker
16,622
439,822
99.9
587
11,304
99.1
10
Cypr
inus
car
pio
Commoncarp
16,575
5167,749
99.9
2,454
6,591
96.4
20
Fund
ulus
not
atus
Blackstripetopminnow
16,510
425,824
99.9
385
16,099
99.1
3
Gam
busia
hol
broo
kiEasternmosquitofish
16,611
765,019
99.9
931
15,745
98.1
3
Lepo
mis
mac
roch
irus
Bluegill
16,489
1834,285
99.9
486
15,580
97.4
7
Pim
epha
les p
rom
elas
Fatheadminnow
16,709
4631
786
.25
1,251
23.6
5
Sem
otilu
s atr
omac
ulat
usCreekchub
16,623
418,304
99.9
267
16,354
98.4
1
JudayCreek(allsitescombined)
Ambl
oplit
es ru
pest
risRo
ck b
ass
16,659
3330,505
98.7
443
16,279
99.8
1
Cato
stom
us c
omm
erso
nii
Whitesucker
16,622
271,367,916
100
20,988
6,644
89.5
14
Cott
us b
aird
iiMottledsculpin
16,529
295
1,296,452
100
18,926
15,873
97.2
3 (Continues)
| 7Methods in Ecology and Evolu onDEINER Et al.
long-range PCR amplification and shotgun sequencing techniques.These results contradict the common assumption that eDNAfrom communities of living fishes inwater is highly degraded (e.g.Bohmann etal., 2014).Our results show instead that some of theeDNAfrommacro-organismscurrently inhabitingawaterbodyre-mains intact at least at the mitochondrial genome size (c. 16kb).Sequencingwholemitogenomesfromwatersamplesisapioneeringwaytonon-invasivelyassesscommunitiesoflivingmacro-organisms.Additionally,itmaymakecharacterizationofmacro-organismeDNArelevantinstudiesofpopulationandconservationgenetics,system-aticsandphylogeography.
This methodological advance alleviates the burden of basingspecies identifications on short-fragment PCR amplicons.Most cur-rent eDNA studies frommacro-organisms base their taxonomic as-signments onDNA fragments of <150base pairs (Port etal., 2016;Valentinietal.,2016).Theamountofinformationinsuchasmallfrag-mentwillalwaysbelimited,andinmanycasesassignmentstothespe-cieslevelarenotpossiblewithoutadditionalinformation.Withwholemitogenomes,entirebarcode regionscanbeexcised fromadatasetandusedfortaxonomicassignment.Forexample,usingthedenovoassemblyapproachwerecoveredfull-lengthmitochondrialgenes(cy-tochromeoxidaseI,cytochromeB,12Sand16S)forthefishspeciesinJudayCreek (e.g.Semotilus atromaculatus),evenwhentheirentiremitochondrialgenomecouldnotbeassembled.Giventheamountofmissingdiagnosticinformationinreferencedatabasesusedformacro-organismtaxonomicassignment(Shawetal.,2016;Trebitz,Hoffman,Grant,Billehus,&Pilgrim,2015),generatingdatawiththismethodwillallowuseofanyregionofthemitogenomeforwhichdataexisttoiden-tifyenvironmentalsequences.Thismethodwillthereforebeinvaluableunlessanduntilthisvoidisfilledwithothermethodssuchasgenomeskimming(Coissac,Hollingsworth,Lavergne,&Taberlet,2016).
While theresults fromour insitu testsarepromising,anumberofquestionsremainabouttheparticularenvironmentalcircumstancesinwhicheDNAisleftintactatthemitogenomelevel.Inthisstudyweusedwatersampled inSeptemberfromasmall third-ordertributarytotheSt.JosephRiverinNorthwesternIndiana,USA.Themeanan-nualdischargeofJudayCreekis0.44m3/ssamples(Oldsetal.,2016).Theaveragetemperatureonthedayofsamplingwas22.6°C(Shirey,Brueseke,Kenny,&Lamberti,2016).Wedidnotcollectothervariablesatthetimeofsamplingasitwasnotourgoaltotesttheseattributeshere,butweencouragefuturestudiestoinvestigateadditionalcon-ditions (e.g. temperature, turbidity, pH), density of individuals, flowconditions,sampletype(e.g.soil,sediment),etc.,thatmayfacilitateorinhibittheamplificationofwholemitogenomes.
Consideration of the species composition is also important.Weobservedthatthedenovoassemblerwasnotabletoassemblesomescaffolds into entiremitogenomes (Figure2).Onepossible explana-tionforthisobservedpatternisthatconservedregionswithhighse-quencesimilarityamongspeciesaredifficult toaccuratelyassemblefromacomplexmixture.Therefore,weexpectthedenovoassemblymethod,whennotguidedbyareferencemappingapproach,willbechallenginginfishcommunitieswhenspeciespairsarecloselyrelatedandsequencesimilarity ishigh.Additionally, fordenovoassembliesSp
ecie
sCo
mm
on n
ame
Refe
renc
e le
ngth
(bp)
N
LRP
map
ped
to re
fere
nce
LRP
de n
ovo
asse
mbl
ed
Uni
quel
y m
appe
d re
ads
Refe
renc
e co
vera
ge (%
)A
vera
ge
sequ
ence
dep
thLo
nges
t sc
affo
ld (b
p)Re
fere
nce
cove
rage
(%)
No.
of
scaf
fold
s
Cypr
inus
car
pio
Commoncarp
16,575
0145,017
100
2,166
16,322
99.7
2
Ethe
osto
ma
caer
uleu
mRainbowdarter
16,588
110,940
100
165
14,429
97.9
3
Ethe
osto
ma
nigr
umJohnnydarter
16,579
891,266,274
100
18,643
6,651
97.7
11
Lepo
mis
cyan
ellu
sGreensunfish
16,485
48309,297
100
4,574
15,882
98.8
3
Lepo
mis
mac
roch
irus
Bluegill
16,489
11
1.7
10
Mic
ropt
erus
dol
omie
uSmallmouthbass
16,488
19286,733
100
4,242
5,915
95.1
12
Mic
ropt
erus
salm
oide
sLargemouthbass
16,484
0232,161
100
3,434
12,879
95.7
8
Onc
orhy
nchu
s myk
issRainbowtrout
16,655
1993,573
100
14,299
16,363
100
3
Rhin
icht
hys a
trat
ulus
Westernblacknosedace
16,651
498,611
93.9
143
16,036
98.6
2
Salm
o tr
utta
Browntrout
16,677
110
5.5
20
Sem
otilu
s atr
omac
ulat
usCreekchub
16,623
562
2,959,545
100
45,213
5,658
95.1
29
TABLE 1 (Continued)
8 | Methods in Ecology and Evolu on DEINER Et al.
| 9Methods in Ecology and Evolu onDEINER Et al.
weobservedthatinsomecaseshighsequencingdepthinhibitedtheconcatenation of multiple shorter scaffolds (Figure2). While downsampling did join smaller scaffolds together more frequently, westill observed the pattern that specieswith the highest read depthtendedtohavethelargestnumberofunassembledscaffolds(Table1,Figure2).Applying long-readsequencingtechnologysuchasPacBioSMRTtechnologytolong-rangeamplifiedproductscouldpotentiallysolvethisproblembyavoidingshortreadsaltogether(Schloss,Jenior,Koumpouras,Westcott,&Highlander,2016).
From the laboratory perspective, there aremany handling stepsthatcouldbeoptimizedtoimprovedetectionofwholemitogenomes.Forexample,thefilterswereextractedwithaChloroform-isoamylDNAextraction,buttheuseofbufferedphenol(pH8.0)inadditiontochlo-roform–isoamyl isknown to increase thequantityofhighmolecularweightDNAduringDNAextractions(Blin&Stafford,1976).Therefore,testingof laboratorymethods fromextraction to librarypreparationmayincreasetheyieldofeDNAsuitableforlong-rangePCR.
WedetectedasmallamountofcontaminationbetweensamplesrunonthesameMiSeqrun(i.e.JudayCreekandthemockcommu-nity).Thelowlevelofcontaminationbetweenlibrariesdidnotresultinhighcoverageofthemitogenomesnorallowdenovoassemblyofcompletemitogenomes in these samples.Additionally, because ourstudydesignintentionallyusedtropicalmarinefishforthemockcom-munity,theycouldeasilybeexcludedfromoccurringinJudayCreek,atemperatefreshwaterhabitat.Wecannotdeterminefromourstudy’sdesignatwhichpointthecontaminationhappenedbecausethelibrar-ies showing the greatest amount of cross contaminationwere alsoprocessed in the laboratory at the same time (i.e.mock communityand Juday Creek samples). The mock community and Juday Creek librarieswerealsopreparedusingtheTruSeqNanoLTSamplePrepKitusingasingleindexstep.Duelindexinghelpstocircumventproblemsassociatedwith“tagjumping,”andwecannotruleoutthisphenome-nonasaproblemforourstudy(Schnell,Bohmann,&Gilbert,2015).Futureapplicationsshouldtakecaretophysicallyandtemporallysep-aratetheprocessingofsamplesthatmaybecomecontaminatedandwerecommendduelindexingsamplestodetectwithgreateraccuracyfalsepositivereadsinlibrariesrunonthesameMiSeqflowcell.
Theprimersdesignedhereweredemonstratedinsilicotopoten-tiallybeusefulfordetectingabroadarrayoffishspeciesoftheclassActinopterygii.Theseprimers couldbemadeevenmore general byadding degenerate bases to sites showing variation in specieswith<95%match at the primer binding region (Appendix S2). Similarly,long-range PCR primers could be designed for other taxonomicgroups,suchasbirds,mammalsandamphibians.Ourlaboratoryandbioinformaticapproachshouldbegenerallyapplicable toanimalmi-tochondrialgenomes.However,giventhecurrentupperlimitstothe
lengthoflong-rangePCRamplifications,themethodwillmakeampli-fyinggenomesfromlargerorganelles,suchaschloroplastsandplantmitochondria,problematic.
Extendingtheuseofourmethodtoresearchfieldslikepopulationgeneticswillrequireadditionalstudies.Forexample,whileoutsidethescopeofthisstudy,thereisthepotentialtophasehaplotypesforgenesorwholemitogenomesfromalignedshortfragments(O’Neil&Emrich,2012).PhasedhaplotypesfromeDNAshotgun-sequenceddatawouldallow for population-level analyses from communities. For example,itmaybecomepossibletoestimatepopulationsizefromeDNAsam-plesusingareverseinferencemethodfrompopulationgeneticequa-tions that estimatemutation rate and haplotype diversity (Wares &Pappalardo,2015).Usingpopulationgenetic theory,at the least, theminimumnumberof individualscanbe inferredfromthehaplotypesandusedtoestimateminimumpopulationsizesthatcontributedtoasampleofeDNA.However,tomakeuseofthistheory,thereisaneedforcontinuedresearchfocusedonparsingoutsequencingnoisefromrealvariationtodetermineintra-specieshaplotypediversitycollectedfromenvironmentalsamplesandhigh-throughoutsequencing(Gómez-Rodríguez,Crampton-Platt,Timmermans,Baselga,&Vogler,2015).
WhileitispromisingthatwecouldidentifySNPsandestimateal-lele frequencies, there remainmany questions about thevalidity ofthese estimates from eDNA. Specifically,we could not confirm ourSNPs from tissues of the actual species and such tests are neededbeforeadoptionofthismethodiswarranted.Untilnow,moststudiesgenerateoperationaltaxonomicunits(OTUs)andsummarizethedataforaspeciesat97%–99%similarity(Hänflingetal.,2016;Oldsetal.,2016),andthushavenotutilizedtheintraspecificlevelvariationpres-entineDNAsamples.ArecentstudybySigsgaardetal.(2016)demon-strated that haplotypediversity is attainable fromwater samples atleast at the single-species level using primers that targeted a smallamplicon (<500bp) inwhale sharks.We expect that future studies applyingourmethodofwholemitochondrialgenomesequencingfromwatersampleswillyieldsimilarresults,butatthecommunityscaleforentiremitogenomes.
The continued advancement of single molecule and long-readtechnologies, such as the Oxford Nanopore MinION (Laszlo etal.,2014),will improve our approach.Here,we used Illumina sequenc-ingwhichrequiredsheeringthemitogenomes,usingsonicationoratransposase-mediatedprocessusedintheNexteralibrarypreparationkit,tofragmentthembeforesequencingandsubsequentlyremappingthese reads toa referencesequenceorconductingdenovoassem-bly.Couplinglong-rangePCRamplificationsandsequencingwithoutfragmentation would avoid many of the problems associated withshort-fragment based assembly or reference mapping. We expectthatlong-readsequencingtechnologies,oncetheyarecosteffective
F IGURE 2 Linearvisualizationforlong-rangePCR-amplifiedfishmitogenomessequencedfromenvironmentalDNAwatersampledinJudayCreek,IN,USA.Speciesaredepictedinalphabeticalorderfromtoptobottomexcludingthetwospecies(Lepomis macrochirus and Salmo trutta)thatdidnotproduceafullmitogenome(Table1).Thenumberedorangebarwithyellowbackgroundisthereferencesequence.Thebluegraphicindicatesthenumberofreadsmappedateachsite(i.e.sequencingdepthinTable1)andeachoftheblackbarsbelowthereferencesequenceareindependentscaffoldsthatweredenovoassembledandsubsequentlymappedtothereference.Scaffoldsconnectedwitharedlineareasinglescaffoldandthelineconnectingthemindicatesagapintheassembly.Gapsinthereferencesequence(indicatedbyaredline)representinsertionscomparedtothedenovoassembledscaffold
10 | Methods in Ecology and Evolu on DEINER Et al.
anderrorratesarereduced,willbecomethemethodofchoiceforse-quencinglong-rangePCRproductsandwillallowpopulationgeneticanalysis of eDNA samples.
ACKNOWLEDGEMENTS
Wewouldliketothankthefollowingindividualsfortissuedonationsthatfacilitatedthiswork:CameronTurner,MikeBruesekeandNathanEvans (University of Notre Dame); we additionally thank DominicChalonerforprovidingtemperaturedataforJudayCreek.WewouldliketothanktheNotreDameGenomics&BioinformaticsCoreFacilityfor theirassistancewith the IlluminaMiSeq.WealsothankScottJ.Emrich (University of Notre Dame) for his assistance in code de-velopment for down sampling our data for de novo assembly. ThisworkwassupportedbytheU.S.DepartmentofDefense’sStrategicEnvironmentalResearchandDevelopmentProgram (RC-2240).Theauthorsdeclarenocompetingfinancialinterests.ThisisapublicationoftheNotreDameEnvironmentalChangeInitiative.
AUTHORS’ CONTRIBUTIONS
Allauthorscontributedtostudydesign;M.A.R.conductedthelabora-torywork;K.D.,M.A.R.,Y.L.,B.P.O.andM.E.P.analysedthedata;andK.D.andM.A.R. led thewritingof themanuscript.All authorscon-tributedcriticallytothedraftsandgavefinalapprovalforpublication.
DATA ACCESSIBILITY
All rawdata associatedwith this studyhavebeendepositedon theNCBI’s Sequence Read Archive (SRA) (www.ncbi.nlm.nih.gov/sra)undertheBioProjectPRJNA317862:SRS2218772(MockCommunity),SRS2218773 (Mesocosm_HE3), SRS2218776 (Mesocosm_HS3) andSRS2218777(Juday_Creek).AllotherintermediateprocesseddatafilesandcodeusedforanalysisareavailablefromDryadDigitalRepository,http://dx.doi.org/10.5061/dryad.q5gg0(Deineretal.,2017).
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the supportinginformationtabforthisarticle.
How to cite this article:DeinerK,RenshawMA,LiY,OldsBP,LodgeDM,PfrenderME.Long-rangePCRallowssequencingofmitochondrialgenomesfromenvironmentalDNA. Methods Ecol Evol. 2017;00:1–11. https://doi.org/10.1111/2041-210X.12836