Posted on Authorea 21 Sep 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160071155.58915559 | This a preprint and has not been peer reviewed. Data may be preliminary. unn title: Running [email protected] ; [email protected] authors: *Corresponding Norway. Oslo, 2 0316 Blindern, 1066 Box P.O. 1 fungal Estensmo eleven H˚avard of F. 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DNA region, of coupled diversity metabarcoding ITS in (HTS), allelic DNA fungal variation the during the investigated sequence are We like species intraspecific often – species. of 18S, of markers over-splitting and effects variable to 16S more the lead like but other assess can markers, species, samples, which DNA related variation, environmental conserved closely While intraspecific from among considerable communities potential. discriminate full complex to its analyzing able limiting for not challenges approach methodological powerful still are a there become has metabarcoding DNA Abstract 2020 21, September 4 3 2 1 Skrede species Estensmo fungal Lena eleven Eva of study DNA case during A variation metabarcoding: sequence intraspecific of influence The auai idvriyCne,Vnela 5 ..Bx91,20,R edn h Netherlands the Leiden, RA 2300, 9517, Box P.O. 55, Vondellaan Center, Biodiversity Naturalis nvriyi Oslo in University Oslo of Sciences University Natural and Center Biodiversity Mathematics Naturalis of Faculty Oslo of University eto o eeisadEouinr ilg Eoee,Dprmn fBocecs nvriyo Oslo, of University Biosciences, of Department (Evogene), Biology Evolutionary and Genetics for Section 1 n H˚avard and Kauserud , altp iest mat N metabarcoding DNA impacts diversity Haplotype 1 1 1 ud Maurice sundy , ud Maurice Sundy , 4 1 usMorgado Luis , 1 usMorgado Luis , 1 1,2 er .Martin-Sanchez M. Pedro , 2 er Martin-Sanchez Pedro , 1 ne Skrede Inger , 3 Inger , 1 , Posted on Authorea 21 Sep 2020 | The copyright holder is the author/funder. All rights reserved. No reuse without permission. | https://doi.org/10.22541/au.160071155.58915559 | This a preprint and has not been peer reviewed. Data may be preliminary. cls 05.Ltr oeeaoaeapoce eedvlpdi re obte itnus ewe PCR between distinguish better and Westcott to 2013; order Edgar in 2010; developed al. were et approaches cluster Caporaso, elaborate 2009; to more based al. was entities), et Later, approach (Schloss, taxonomic 2015). first threshold Schloss biological similarity One for data sequence approximations analyses. 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(Taberlet, metabarcoding communities DNA i.e. microbial markers study amplified to of (HTS) sequencing throughput High Introduction species-level approach to units needed basic is as step used words: clustering be Key extra not an should pos- ASVs) but PCR (and (or intraspecific to analyses, of haplotypes community due resolution. presence that likely fungal The suggest were ITS-based region. ITS2 frequencies, rDNA in detected in low the haplotypes variation in in additional intragenomic) variation few occurring intragenomic sibly a often or of haplotypes, errors some exception haplo- sequencing extra between included the and These correspondence with one, high approach. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary. eune noeIlmn ie Ilmn,SnDeo A S)ln ih2 with lane USA) and CA, Assay PhiX Diego, 20% BR San with DNA spiked (Illumina, ds adapters, MiSeq Qubit Illumina with with Illumina barcoded measured one were was in magnetic libraries concentration two XP sequenced DNA The AMPure normalization the Agencourt Technologies). to using and (Life prior purified kit AS) gel 20 and Meszansky in concentrated agarose (Nerliens eluted pooled, 2% were beads and a library (Invitrogen) on each Kit electrophoresis within Plate by products Normalization controlled SequalPrep was the products using PCR of quality The 72 at extension s, 30 95 at activation ng/ 5-10 and nooet i T2hpoye e pce Tbe1,ietfigattl4 altpsfo h Sanger the from haplotypes 45 total population. local a of the identifying dephased in level 1), were present high (Table were sequences variation or species ITS2 except sequence templates) The per ITS2 species, sequences, multiple haplotypes all interpret. low-quality For fruit (generating ITS2 to in 16 six dataset. bodies hard resulted to to chromatograms or fruit 6 one amplify to from the not into leading ranging inside did bodies, indels, growing either fruit of fungi 176 bodies heterozygosity of fruit fungicolous out remaining to 151 The due for species. were sequences datasets per Sanger haplotype ITS2 bodies two quality the high obtained from We sequences the 2016). last, al. 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All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary. ,Asa ,Ceh P aedH ta.21.Srcueadfnto ftegoa osi microbiome. topsoil global the of function and Structure 2018. al. et Bengtsson-Palme H, PM, Harend Bodegom LP, NA, Soudzilovskaia Coelho JL, S, Anderson Anslan SK, Forslund All J, F, manuscript. Hildebrand M, the Bahram wrote HK and SM ELE, References LM. and ELE manuscript. sequencing. SM, Sanger the HK, approved and processed preparation and from SM library edited contributions sampling. HTS authors performed performed with HK ELE data and and SM SM analyzed research. DNA. the extracted conceptualized and and samples designed ELE and SM HK, Repository Digital contributions Dryad Author the at available are codes and dataset HTS (http://dx.doi.org/issakka/its NCBI. in deposited are sequences Sanger Accessibility Data interest. competing no declare authors University The the at Biosciences of Interests Department the Competing and supported 254746) financially No was (grant research Norway Oslo. The of of organization. Council sampling with Research help the for by Kuhmo Mets in from Center Meriruoko Research Park Ari Helo, Teppo acknowledge We will classification, we taxonomic direction, reliable this more Acknowledgements towards tools. provide head bioinformatics to To suitable regions 2018). to different al. addition of coverage et in higher Tedersoo, promising with 2018; 16S, databases are al. for require Nanopore) et bp (Kennedy, Oxford samp- 500-1500 environmental resolution PacBio, (e.g. most taxonomic (e.g. barcodes from datasets longer technologies taxonomic multi-locus generate improve sequencing generate to to to generation position required third a often Alternatively, in when are not les. species markers still resolve related DNA to are closely approach we independent Yet, powerful separate multiple resolution. a to marker, is metabarcoding and DNA DNA da- populations single of with natural targeting limitations error-correction in variation and the allelic HTS Given the on communities. reflects based complex extent metabarcoding, large DNA a that different to conclude across da2, we distributions results, same our the to frequent show According most 2018). largely The different al. they data. against et 16S and robust and (Botnen, pattern ITS highly treatments community line for are the data In both drive patterns 99%, 2018). diversity ASVs) to Martiny results similarity (or beta and studies sequence comparable OTUs that 85% (Glassman In demonstrated that from clusters aims. ranging (2018) shown sequence levels, study al with clustering representing been et the OTUs species, Botnen previously on or this, 11 ASVs depends has with the using this it obtained representing of species- turnover), be approach OTUs importance (community results can to 13 The diversity Our correction haplotypes. obtained variation. error beta species two after we intraspecific emphasizing between by needed with haplotypes, separate represented is markers our step may species variable clustering clustering mutation two for a After pair approach marker, case reasonable resolution. base ITS the the variable a level single not as the is a however haplotypes) in this as when is that Indeed, to show 18S, This referred 2017). genera. and largely al. even 16S study et or our like (Callahan, (in markers, analyses ASVs conserved community term for microbial the previously in use been to units advocated has basic been region in recently ITS 2003), has Schumacher It the and Kauserud in 2002; results. variation Schumacher our and sequence with (Kauserud species, scales line spatial target broader the across reported of some For Basidiomycota. lseig n ilb aeaalbeuo publication. upon available made be will and clustering) 6 > hliu n egl ´roy rmteFriendship the V´arkonyi from Gergely and ¨ ahallitus 0 pfrISad60b o O)adimprove and COI) for bp 650 and ITS for bp 700 Posted on Authorea 21 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary. end G ln C ogZ 08 rbn rms esspromnei ogrra uglmetabar- fungal read longer in performance versus promise Probing 217:973-976. 2018. phytologist Z. New Song coding. LC, fungus Cline wood-decay PG, pioneer the Kennedy of Phellinus structure fungus population 95:416-425. local decay Mycologia and 80:597-606. abietinum. wood Regional Trichaptum botanique 2003. endangered de T. 454- Schumacher the canadien H, by Journal of Kauserud region–evaluation botany. structure of ITS2 journal Population fungal Canadian 2002. (Basidiomycota). the T. nigrolimitatus 82:666-677. Schumacher amplify ecology microbiology H, to FEMS Kauserud primers communities. natural New and 2012. artificial of KE. sequencing Clemmensen 9:e87624. M, the ONE of Durling PLOS Characterization primers. 2014. B specific C. K, eukaryote Troedsson Ihrmark universal EM, designing Thompson for I, sequencing gene Jonassen next-generation rRNA A, of 18S Lanzen years K, Lekang ten K, age: Hadziavdic of 17:333-351. Coming Genetics 2016. variants Reviews WR. sequence Nature McCombie exact technologies. JD, of McPherson use S, to Goodwin robust 3. are MSphere the patterns units. to ecological taxonomic basidiomycetes–application Broadscale operational 2018. for versus JB. specificity Martiny 2:113-118. enhanced SI, Ecology Glassman with Molecular primers rusts. and ITS mycorrhizae 1993. of TD. identification Bruns methods Nature M, reads. Gardes amplicon microbial from sequences OTU 3:613- accurate 10:996. highly Evolution UPARSE: of and 2013. metabarcoding Ecology RC. soup: Edgar in Biodiversity 2012. Methods Z. biomonitoring. Ding and sequencing C, Yang assessment community C, 623. biodiversity Ye high-throughput Pe˜na rapid AG, X, Wang of for N, BC, analysis arthropods Fierer Emerson Y, allows EK, Ji WY, QIIME Costello Douglas FD, 2010. Bushman al. 7:335-336. K, et methods Nature Bittinger JI, High- data. J, Gordon 2019. Stombaugh MK. JK, J, Dougherty Goodrich SK, Kuczynski Nucleic McGill JG, resolution. AS, single-nucleotide Caporaso with Gulati gene CM, rRNA Theriot 16S 47:e103. full-length S, Research the Acids Oh of C, sequencing Heiner amplicon high-resolution throughput J, DADA2: 13:581. Wong 2016. methods SP. BJ, Nature Holmes Callahan data. AJA, amplicon Johnson AW, Illumina Han from MJ, inference Rosen sample PJ, 11:2639-2643. McMurdie software journal taxonomic BJ, ISME unix-inspired operational Callahan replace The a should analysis. variants Obitools: data sequence 2016. marker-gene Exact 2017. E. in SP. units Coissac Holmes P, PJ, McMurdie Taberlet 16:176-182. BJ, Resources Y, Callahan on Ecology Bras effect Molecular Le metabarcoding. little A, DNA has Bonin for threshold package C, 18:1064-1076. clustering DNA Resources Mercier Sequence Ecology as F, 2018. Molecular Boyer ITS2 structure. H. versus community Kauserud ITS1 microbial R, of 2013. Halvorsen recovery H. the ML, Kauserud Davey PM, SS, 13:218-224. Kirk Botnen Resources K, Ecology Abarenkov Molecular RH, fungi. surrogate Nilsson for with S, metabarcodes compared Kumar composition R, arthropods stand of Blaalid forest metabarcoding 101:313-323. detail: to Indicators the response Ecological in biodiversity. is biodiversity devil of of The measures 2019. measure DW. sensitive Yu Y-Q, a Ji provides J, Forster C, Bruce N, Barsoum 560:233-237. Nature dkrI rzMrie ,FiegH uatv ,ShnkJ ti ,SeldJ Brandstr J, Stenlid Y, Strid J, Schenck A, Kubartova H, Friberg K, Cruz-Martinez I, ¨ odeker 7 ¨ om- Posted on Authorea 21 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary. tcerntE oblB.19.Txnmcnt:apaefrDADArascainad1SrRNA 44:846-849. 16S Microbiol Evol and Syst reassociation J DNA-DNA Int bacteriology. for in place definition quercus a species present a note: the from Taxonomic in roots 1994. analysis ectomycorrhizal BM. sequence pooled Goebel and E, sporocarps Stackebrandt ectomycorrhi- of of sequencing variation ITS rDNA 18:15-22. intra-sporocarp Mycorrhiza by and woodland. assessed 109:6241-6246. Intra-specific as PNAS ribosomal 2007. fungi. Nuclear fungi DM. for 2012. Rizzo zal marker W. GW, barcode Chen Douhan DNA CA, ME, universal Levesque Smith a JL, as Spouge region V, (ITS) Robert spacer S, transcribed internal Huhndorf KA, Seifert CL, Schoch 75:7537- microbiology environmental Parks and BB, Applied communities. Oakley microbial RA, 7541. comparing Lesniewski soft- and community-supported EB, describing platform-independent, Hollister for open-source, Evolution M, ware mothur: Hartmann Introducing Biology 2009. JR, Molecular CJ. Hall Robinson sets. T, DH, Ryabin data SL, for large Westcott PD, of tool Schloss analysis source polymorphism open sequence versatile DNA Sanchez-Gracia SE, 34:3299-3302. 6: a Ramos-Onsins P, DnaSP VSEARCH: Librado 2017. S, 2016. Guirao-Rico A. JC, Mah´e F. Sanchez-DelBarrio A, Ferrer-Mata C, J, Rozas Quince B, 4:e2409v2401. Nichols Preprints PeerJ T, Statistical metagenomics. Flouri for Foundation T, R computing. Rognes statistical the for on environment http://www.r-project.org/index.html. dependent and Austria. language highly Vienna, A is Computing, R: data (2019). Team community 2019. Core fungal C. R Vacher soil J, 41:23-33. and Vallance ecology I, plant Fungal Lesur of pipeline. A, metabarcoding accuracy molecular Gautier L, the for Fauchery matters: implications V, in Bioinformatics its Edel-Hermann variability V, and Laval ITS Bu´ee databases M, C, Intraspecific sequence Pauvert 2008. international 4:EBO.S653. K-H. the Bioinformatics Acids Larsson in Evolutionary Nucleic N, identification. expressed Hallenberg species DNA. as M, plant fungi Ryberg kingdom weight E, the molecular Kristiansson high RH, of Nilsson isolation Rapid 1980. EMBnet. 8:4321-4325. reads. WF. Research sequencing Thompson high-throughput MG, from Murray sequences adapter removes 17:10-12. Cutadapt journal 2011. high-resolution and M. highly-scalable v2: Martin Swarm 2015. 454 M. 3:e1420. Dunthorn Employing C, PeerJ Vargas clustering. 2013. de C, amplicon H. region Quince T, rDNA Kauserud Rognes spacer F, T, Mahe transcribed 3:1751-1764. Schumacher internal Evolution M, the and in Davey Ecology divergence fungi. R, intragenomic in Nilsson 103:731-740. reveal Mycologia to Henrik pyrosequencing Laetiporus. relati- T, genus phylogenetic amplicon Carlsen in obscures units region DL, taxonomic rDNA Lindner ITS operational the of in estimates variation inflates Intragenomic and markers–a 2011. onships amplified MT. of Banik sequencing DL, high-throughput Lindner by Rosendahl T, analysis 199:288-299. Pennanen community phytologist U, Fungal New Koljalg 2013. R, guide. al. Kjoller in user’s T, et Methods Carlsen J, K, construction. Stenlid Abarenkov L, network S, Tedersoo RH, haplotype Nilsson for BD, Lindahl software full-feature 6:1110-1116. popart: Evolution identification 2015. and molecular D. Ecology the Bryant for methods JW, Kjoller web-based K, Leigh providing 166:1063-1068. Hoiland database S, phytologist a Erland New U, UNITE: fungi. Eberhardt 2005. ectomycorrhizal IJ, al. of Alexander et RH, E, Nilsson Larsson K, R, Abarenkov KH, Larsson U, Koljalg 8 Posted on Authorea 21 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary. trifuga cen- Phlebia ium separ- Gloeophyllum rosea Fomitopsis cola pini- Fomitopsis serialis Antrodia ica lappon- Amylocystis Specimen Species approach. HTS either by and identified haplotypes Sanger common additional between dikaryotic include and each (Hap.) overlap HTS haplotypes from and Total the sequence variants. Sanger sequence is ITS2 both amplicon the from pair) for to haplotypes stands (base correspond ASVs and length sequences individual dephased Sequence (n+n) of shown. number alignments, are sequence approaches both RNA from ribosomal fungal 1. of 18:315-322. Table applications sequencing and direct methods and to Amplification guide Tables 1990. a J. protocols: Taylor PCR S, phylogenetics. Lee for genes 3:e1487. T, PeerJ Bruns units. TJ, taxonomic White assigning operational for to methods conse- sequences reference-based its outperform gene methods and rRNA clustering novo 16S genomes De bacterial 2015. PD. 8:e57923-e57923. in Schloss SL, ONE gene Westcott PLOS rRNA analyses. 16S community the bacterial eukaryotes: for of other quences variability and The fungi 2013. 217:1370-1385. of P. 346:1256688. phytologist Vˇetrovsk´y metabarcoding Science Baldrian New PacBio T, fungi. perspectives. 2018. Thu soil and AM, S. of biases Vasco-Palacios geography Anslan LV, errors, and A, Ruiz diversity Tooming-Klunderud R, Global L, Wijesundera Tedersoo 2014. NS, Yorou al. U, et Koljalg A, S, Suija Polme PQ, M, Bahram 21:2045-2050. L, biodiversity Tedersoo Ecology next-generation Towards Molecular 2012. E. metabarcoding. Willerslev DNA C, monito- using Brochmann and assessment F, research Pompanon biodiversity E, Coissac for P, DNA: Taberlet Environmental 2018. 2017. Press. L. M. University Zinger Bunce Oxford E, ring: ES, Coissac A, Harvey environment. Bonin marine SJ, P, tropical Taberlet Newman a in TE, life Berry of tree 7:1-11. JD, the Reports across DiBattista Scientific metabarcoding R, eDNA: with Bernasconi biomonitoring MJ, Ecosystem Huggett M, Stat oprsno agradHSsqecs nyseiesfrwihsqecswr obtained were sequences which for specimens Only sequences. HTS and Sanger of Comparison 6263 5,6 044 6 4 8 5 9 10 8 4 355,067 5 8 1 429,990 6 4 168,991 7 1 238,143 3 2 178,787 0 5 66,364 1 5 5 32 5 5 4 32 276 5 1 30 271 3 16 32 277 0 16 32 229 15 18 188 16 308 16 9 (bp) length Sequence a sequences Dephased quences se- Sanger sites Polymorphic quences se- Sanger 9 a.RasPolymorphic Reads Hap. une SsAV ASVs ASVs ASVs quences se- Sanger

sites Hap. Total Haplo- types Posted on Authorea 21 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary. o euneaudnei h T aaie 0tmsblwwa ol eepce rmasnl leein allele single a from expected be very would what with (Hap below blue occurring haplotypes times and haplotypes of 10 dataset, indicate naming indicates i.e. The arrows HTS data color Red from HTS population. Green the the haplotypes sequencing. in yellow step. Sanger abundance HTS, mutational by sequence low and one detected sequencing represent only Sanger were dash by haplotypes each both and detected haplotype haplotypes one represents circle Each y-axis). 2. on Figure HTS and x-axis on sequencing (Sanger 1. Figure Figures inum abiet- Trichaptum caesia Postia viticola Phellinus itatus grolim- ni- Phellopilus cus neofus- ferrugi- Specimen Phellinus Species altp ewrsdslyn h ee fitapcfi aito nIS o h 1fna species. fungal 11 the for ITS2 in variation intraspecific of level the displaying networks Haplotype iltsoigtecrepnec fhpoye nrltv bnac costetodatasets two the across abundance relative in haplotypes of correspondence the showing Biplot 5283 4,6 8 4 5 6 4 11 5 7 148,066 10 7 5 191,483 4 120,306 8 5 46,677 5 5 3 178,295 5 7 2 6 30 4 20 268 9 32 4 232 15 281 12 5 10 296 16 32 6 282 16

(bp) length Sequence a quences se- Sanger

oHap to 1 quences se- Sanger 10 5 olw al S1. Table follows 65) une SsAV ASVs ASVs ASVs quences se- Sanger Total Haplo- types Posted on Authorea 21 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary.

Prop. abundance HTS

0.0 0.2 0.4 0.6 0.8 1.0 0.0 p R 2 = = 7.6e−15 0.613 0.2 Prop. abundance Sangersequencing 0.4 11 0.6 0.8 1.0 Posted on Authorea 21 Sep 2020 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.160071155.58915559 — This a preprint and has not been peer reviewed. Data may be preliminary. Hap_19 Fomitopsis rosea Amylocystis lapponica Hap_56 Hap_36 Phellopilus nigrolimitatus Phellopilus Postia caesia Hap_48 Phellinus ferrogineofuscus Hap_22 Hap_20 Hap_57 Hap_18 Hap_15 Hap_54 Hap_34 Hap_1 Hap_38 Hap_16 Hap_55 Hap_21 Hap_37 Hap_41 Hap_43 Hap_65 Hap_17 Hap_5 Hap_33 Hap_60 Gloeophyllum sepiarium Hap_59 Hap_24 Hap_47 Antrodia serialis Trichaptum abietinum Hap_3 Hap_35 Hap_28 Hap_25 Hap_26 Hap_23 12 Hap_2 Hap_58 Hap_46 Hap_64 Hap_4 Hap_42 Hap_27 Hap_63 Hap_30 Hap_9 Hap_61 Hap_40 Fomitopsis pinicola Hap_62 Hap_29 Hap_10 Phlebia centrifuga Hap_44 Hap_39 Hap_1 Hap_13 Hap_52 Hap_6 Hap_31 Hap_45 1 Phellinus viticola Hap_8 Hap_49 Hap_50 Hap_51 Hap_12 Hap_14 Hap_7 Hap_32 Hap_53