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

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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)

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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

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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