Sequencing the orthologs of human autosomal forensic short tandem repeats provides individual- and species-level identification in African great apes.
Bonobo
Chimpanzee
Gorilla
Individual identification
Massively parallel sequencing
Short-tandem repeat (STR)
Single nucleotide polymorphism (SNP)
Verogen ForenSeq™ DNA Signature Prep kit
Journal
BMC ecology and evolution
ISSN: 2730-7182
Titre abrégé: BMC Ecol Evol
Pays: England
ID NLM: 101775613
Informations de publication
Date de publication:
31 Oct 2024
31 Oct 2024
Historique:
received:
29
11
2023
accepted:
17
10
2024
medline:
1
11
2024
pubmed:
1
11
2024
entrez:
1
11
2024
Statut:
epublish
Résumé
Great apes are a global conservation concern, with anthropogenic pressures threatening their survival. Genetic analysis can be used to assess the effects of reduced population sizes and the effectiveness of conservation measures. In humans, autosomal short tandem repeats (aSTRs) are widely used in population genetics and for forensic individual identification and kinship testing. Traditionally, genotyping is length-based via capillary electrophoresis (CE), but there is an increasing move to direct analysis by massively parallel sequencing (MPS). An example is the ForenSeq DNA Signature Prep Kit, which amplifies multiple loci including 27 aSTRs, prior to sequencing via Illumina technology. Here we assess the applicability of this human-based kit in African great apes. We ask whether cross-species genotyping of the orthologs of these loci can provide both individual and (sub)species identification. The ForenSeq kit was used to amplify and sequence aSTRs in 52 individuals (14 chimpanzees; 4 bonobos; 16 western lowland, 6 eastern lowland, and 12 mountain gorillas). The orthologs of 24/27 human aSTRs amplified across species, and a core set of thirteen loci could be genotyped in all individuals. Genotypes were individually and (sub)species identifying. Both allelic diversity and the power to discriminate (sub)species were greater when considering STR sequences rather than allele lengths. Comparing human and African great-ape STR sequences with an orangutan outgroup showed general conservation of repeat types and allele size ranges. Variation in repeat array structures and a weak relationship with the known phylogeny suggests stochastic origins of mutations giving rise to diverse imperfect repeat arrays. Interruptions within long repeat arrays in African great apes do not appear to reduce allelic diversity. Orthologs of most human aSTRs in the ForenSeq DNA Signature Prep Kit can be analysed in African great apes. Primer redesign would reduce observed variability in amplification across some loci. MPS of the orthologs of human loci provides better resolution for both individual and (sub)species identification in great apes than standard CE-based approaches, and has the further advantage that there is no need to limit the number and size ranges of analysed loci.
Sections du résumé
BACKGROUND
BACKGROUND
Great apes are a global conservation concern, with anthropogenic pressures threatening their survival. Genetic analysis can be used to assess the effects of reduced population sizes and the effectiveness of conservation measures. In humans, autosomal short tandem repeats (aSTRs) are widely used in population genetics and for forensic individual identification and kinship testing. Traditionally, genotyping is length-based via capillary electrophoresis (CE), but there is an increasing move to direct analysis by massively parallel sequencing (MPS). An example is the ForenSeq DNA Signature Prep Kit, which amplifies multiple loci including 27 aSTRs, prior to sequencing via Illumina technology. Here we assess the applicability of this human-based kit in African great apes. We ask whether cross-species genotyping of the orthologs of these loci can provide both individual and (sub)species identification.
RESULTS
RESULTS
The ForenSeq kit was used to amplify and sequence aSTRs in 52 individuals (14 chimpanzees; 4 bonobos; 16 western lowland, 6 eastern lowland, and 12 mountain gorillas). The orthologs of 24/27 human aSTRs amplified across species, and a core set of thirteen loci could be genotyped in all individuals. Genotypes were individually and (sub)species identifying. Both allelic diversity and the power to discriminate (sub)species were greater when considering STR sequences rather than allele lengths. Comparing human and African great-ape STR sequences with an orangutan outgroup showed general conservation of repeat types and allele size ranges. Variation in repeat array structures and a weak relationship with the known phylogeny suggests stochastic origins of mutations giving rise to diverse imperfect repeat arrays. Interruptions within long repeat arrays in African great apes do not appear to reduce allelic diversity.
CONCLUSIONS
CONCLUSIONS
Orthologs of most human aSTRs in the ForenSeq DNA Signature Prep Kit can be analysed in African great apes. Primer redesign would reduce observed variability in amplification across some loci. MPS of the orthologs of human loci provides better resolution for both individual and (sub)species identification in great apes than standard CE-based approaches, and has the further advantage that there is no need to limit the number and size ranges of analysed loci.
Identifiants
pubmed: 39482599
doi: 10.1186/s12862-024-02324-0
pii: 10.1186/s12862-024-02324-0
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
134Subventions
Organisme : Natural Environment Research Council
ID : NE/L002493/1
Organisme : Natural Environment Research Council
ID : NE/L002493/1
Organisme : Natural Environment Research Council
ID : NE/L002493/1
Informations de copyright
© 2024. The Author(s).
Références
Maxwell SL, Fuller RA, Brooks TM, Watson JE. Biodiversity: The ravages of guns, nets and bulldozers. Nature. 2016;536(7615):143–5.
pubmed: 27510207
doi: 10.1038/536143a
Williams PH, Burgess ND, Rahbek C. Flagship species, ecological complementarity and conserving the diversity of mammals and birds in sub-Saharan Africa. Animal Conserv. 2000;3:249–60.
Kuhlwilm M, de Manuel M, Nater A, Greminger MP, Krutzen M, Marques-Bonet T. Evolution and demography of the great apes. Curr Opin Genet Dev. 2016;41:124–9.
pubmed: 27716526
doi: 10.1016/j.gde.2016.09.005
Rivers MC, Brummitt NA, Ludhadha EN, Meagher TR. Do species conservation assessments capture genetic diversity? Global Ecol Conserv. 2014;2:81–7.
doi: 10.1016/j.gecco.2014.08.005
Ouborg NJ, Pertoldi C, Loeschcke V, Bijlsma RK, Hedrick PW. Conservation genetics in transition to conservation genomics. Trends Genet. 2010;26(4):177–87.
pubmed: 20227782
doi: 10.1016/j.tig.2010.01.001
Keller LF, Waller DM. Inbreeding effects in wild populations. Trends Ecol Evol. 2002;17:230–41.
doi: 10.1016/S0169-5347(02)02489-8
Xue Y, Prado-Martinez J, Sudmant PH, Narasimhan V, Ayub Q, Szpak M, Frandsen P, Chen Y, Yngvadottir B, Cooper DN, et al. Mountain gorilla genomes reveal the impact of long-term population decline and inbreeding. Science. 2015;348(6231):242–5.
pubmed: 25859046
pmcid: 4668944
doi: 10.1126/science.aaa3952
Jobling MA, Hollox EJ, Hurles ME, Kivisild T, Tyler-Smith C. Human evolutionary genetics. 2nd ed. New York and London: Garland Science; 2014.
Prado-Martinez J, Sudmant PH, Kidd JM, Li H, Kelley JL, Lorente-Galdos B, Veeramah KR, Woerner AE, O’Connor TD, Santpere G, et al. Great ape genetic diversity and population history. Nature. 2013;499(7459):471–5.
pubmed: 23823723
pmcid: 3822165
doi: 10.1038/nature12228
Vigilant L, Guschanski K. Using genetics to understand the dynamics of wild primate populations. Primates. 2009;50(2):105–20.
pubmed: 19172380
doi: 10.1007/s10329-008-0124-z
Allendorf FW. Genetics and the conservation of natural populations: allozymes to genomes. Mol Ecol. 2017;26(2):420–30.
pubmed: 27933683
doi: 10.1111/mec.13948
Jobling MA, Gill P. Encoded evidence: DNA in forensic analysis. Nat Rev Genet. 2004;5:739–51.
pubmed: 15510165
doi: 10.1038/nrg1455
Linacre A. Animal Forensic Genetics Genes (Basel). 2021;12(4):515.
pubmed: 33916063
doi: 10.3390/genes12040515
Wall JD. Great ape genomics. ILAR J. 2013;54(2):82–90.
pubmed: 24174434
pmcid: 3814392
doi: 10.1093/ilar/ilt048
Thakur M, Chandra K, Sahajpal V, Samanta A, Sharma A, Mitra A. Functional validation of human-specific PowerPlex((R)) 21 System (Promega, USA) in chimpanzee (Pan troglodytes). BMC Res Notes. 2018;11(1):695.
pubmed: 30285836
pmcid: 6171178
doi: 10.1186/s13104-018-3803-x
Singh A, Sahajpal V, Thakur M, Sharma LK, Chandra K, Bhandari D, Sharma A. Applicability of human-specific STR systems, GlobalFiler PCR Amplification Kit, Investigator 24plex QS Kit, and PowerPlex(R) Fusion 6C in chimpanzee (Pan troglodytes). BMC Res Notes. 2021;14(1):212.
pubmed: 34051836
pmcid: 8164790
doi: 10.1186/s13104-021-05632-6
Kwong M, Pemberton TJ. Sequence differences at orthologous microsatellites inflate estimates of human-chimpanzee differentiation. BMC Genomics. 2014;15:990.
pubmed: 25407736
pmcid: 4253012
doi: 10.1186/1471-2164-15-990
Gettings KB, Kiesler KM, Faith SA, Montano E, Baker CH, Young BA, Guerrieri RA, Vallone PM. Sequence variation of 22 autosomal STR loci detected by next generation sequencing. Forensic Sci Int Genet. 2016;21:15–21.
pubmed: 26701720
doi: 10.1016/j.fsigen.2015.11.005
Beasley J, Shorrock G, Neumann R, May CA, Wetton JH. Massively parallel sequencing and capillary electrophoresis of a novel panel of falcon STRs: Concordance with minisatellite DNA profiles from historical wildlife crime. Forensic Sci Int Genet. 2021;54:102550.
Churchill JD, Schmedes SE, King JL, Budowle B. Evaluation of the Illumina® Beta Version ForenSeq DNA Signature Prep Kit for use in genetic profiling. Forensic Sci Int Genet. 2016;20:20–9.
pubmed: 26433485
doi: 10.1016/j.fsigen.2015.09.009
Just RS, Moreno LI, Smerick JB, Irwin JA. Performance and concordance of the ForenSeq system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens. Forensic Sci Int Genet. 2017;28:1–9.
pubmed: 28126691
doi: 10.1016/j.fsigen.2017.01.001
Kalinowski ST, Wagner AP, Taper ML. ML-Relate: a computer program for maximum likelihood estimation of relatedness and relationship. Mol Ecol Notes. 2006;6:576–9.
doi: 10.1111/j.1471-8286.2006.01256.x
Makova KD, Pickett BD, Harris RS, Hartley GA, Cechova M, Pal K, Nurk S, Yoo D, Li Q, Hebbar P et al. The complete sequence and comparative analysis of ape sex chromosomes. Nature. 2024;630(8016):401–11.
Sullivan KM, Mannucci A, Kimpton CP, Gill P. A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. Biotechniques. 1993;15(4):636–8 640–631.
pubmed: 8251166
Khubrani YM, Hallast P, Jobling MA, Wetton JH. Massively parallel sequencing of autosomal STRs and identity-informative SNPs highlights consanguinity in Saudi Arabia. Forensic Sci Int Genet. 2019;43:102164.
pubmed: 31585345
doi: 10.1016/j.fsigen.2019.102164
Sun JX, Helgason A, Masson G, Ebenesersdottir SS, Li H, Mallick S, Gnerre S, Patterson N, Kong A, Reich D, et al. A direct characterization of human mutation based on microsatellites. Nat Genet. 2012;44(10):1161–5.
pubmed: 22922873
doi: 10.1038/ng.2398
Barbara T, Palma-Silva C, Paggi GM, Bered F, Fay MF, Lexer C. Cross-species transfer of nuclear microsatellite markers: potential and limitations. Mol Ecol. 2007;16(18):3759–67.
pubmed: 17850543
doi: 10.1111/j.1365-294X.2007.03439.x
Maduna SN, Rossouw C, Roodt-Wilding R. Bester-van der Merwe AE: Microsatellite cross-species amplification and utility in southern African elasmobranchs: A valuable resource for fisheries management and conservation. BMC Res Notes. 2014;7:352.
pubmed: 24915745
doi: 10.1186/1756-0500-7-352
FitzSimmons NN, Moritz C, Moore SS. Conservation and dynamics of microsatellite loci over 300 million years of marine turtle evolution. Mol Biol Evol. 1995;12(3):432–40.
pubmed: 7739385
Miles LG, Isberg SR, Glenn TC, Lance SL, Dalzell P, Thomson PC, Moran C. A genetic linkage map for the saltwater crocodile (Crocodylus porosus). BMC Genomics. 2009;10:339.
pubmed: 19640266
pmcid: 2907706
doi: 10.1186/1471-2164-10-339
Primmer CR, Painter JN, Koskinen MT, Palo JU, Merilä J. Factors affecting avian cross-species microsatellite amplification. J Avian Biol. 2005;36(4):348–60.
doi: 10.1111/j.0908-8857.2005.03465.x
Blanquer-Maumont A, Crouau-Roy B. Polymorphism, monomorphism, and sequences in conserved microsatellites in primate species. J Mol Evol. 1995;41(4):492–7.
pubmed: 7563137
doi: 10.1007/BF00160321
Brohede J, Ellegren H. Microsatellite evolution: polarity of substitutions within repeats and neutrality of flanking sequences. Proc Biol Sci. 1999;266(1421):825–33.
pubmed: 10343406
pmcid: 1689914
doi: 10.1098/rspb.1999.0712
Clisson I, Lathuilliere M, Crouau-Roy B. Conservation and evolution of microsatellite loci in primate taxa. Am J Primatol. 2000;50(3):205–14.
pubmed: 10711534
doi: 10.1002/(SICI)1098-2345(200003)50:3<205::AID-AJP3>3.0.CO;2-Y
Gugerli F, Brodbeck S, Holderegger R. Insertions–deletions in a microsatellite flanking region may be resolved by variation in stuttering patterns. Plant Mol Biol Report. 2008;26:255–62.
doi: 10.1007/s11105-008-0034-7
Estoup A, Jarne P, Cornuet JM. Homoplasy and mutation model at microsatellite loci and their consequences for population genetics analysis. Mol Ecol. 2002;11(9):1591–604.
pubmed: 12207711
doi: 10.1046/j.1365-294X.2002.01576.x
Salado I, Fernandez-Gil A, Vila C, Leonard JA. Automated genotyping of microsatellite loci from feces with high throughput sequences. PLoS ONE. 2021;16(10):e0258906.
pubmed: 34695152
doi: 10.1371/journal.pone.0258906
Bonnin N, Piel AK, Brown RP, Li Y, Connell AJ, Avitto AN, Boubli JP, Chitayat A, Giles J, Gundlapally MS, et al. Barriers to chimpanzee gene flow at the south-east edge of their distribution. Mol Ecol. 2023;32(14):3842–58.
pubmed: 37277946
doi: 10.1111/mec.16986
Wroblewski EE, Guethlein LA, Anderson AG, Liu W, Li Y, Heisel SE, Connell AJ, Ndjango JN, Bertolani P, Hart JA, et al. Malaria-driven adaptation of MHC class I in wild bonobo populations. Nat Commun. 2023;14(1):1033.
pubmed: 36823144
doi: 10.1038/s41467-023-36623-9
Barbian HJ, Connell AJ, Avitto AN, Russell RM, Smith AG, Gundlapally MS, Shazad AL, Li Y, Bibollet-Ruche F, Wroblewski EE, et al. CHIIMP: An automated high-throughput microsatellite genotyping platform reveals greater allelic diversity in wild chimpanzees. Ecol Evol. 2018;8(16):7946–63.
pubmed: 30250675
pmcid: 6145012
doi: 10.1002/ece3.4302
Prufer K, Munch K, Hellmann I, Akagi K, Miller JR, Walenz B, Koren S, Sutton G, Kodira C, Winer R, et al. The bonobo genome compared with the chimpanzee and human genomes. Nature. 2012;486(7404):527–31.
pubmed: 22722832
pmcid: 3498939
doi: 10.1038/nature11128
Scally A, Dutheil JY, Hillier LW, Jordan GE, Goodhead I, Herrero J, Hobolth A, Lappalainen T, Mailund T, Marques-Bonet T, et al. Insights into hominid evolution from the gorilla genome sequence. Nature. 2012;483(7388):169–75.
pubmed: 22398555
pmcid: 3303130
doi: 10.1038/nature10842
Darby BJ, Erickson SF, Hervey SD, Ellis-Felege SN. Digital fragment analysis of short tandem repeats by high-throughput amplicon sequencing. Ecol Evol. 2016;6(13):4502–12.
pubmed: 27386092
pmcid: 4930997
doi: 10.1002/ece3.2221
Barrett KG, Amaral G, Elphinstone M, McAdie ML, Davis CS, Janes JK, Carnio J, Moehrenschlager A, Gorrell JC. Genetic management on the brink of extinction: sequencing microsatellites does not improve estimates of inbreeding in wild and captive Vancouver Island marmots (Marmota vancouverensis). Conserv Genet. 2022;23(2):417–28.
pubmed: 35401067
pmcid: 8948115
doi: 10.1007/s10592-022-01429-7
Willems T, Zielinski D, Yuan J, Gordon A, Gymrek M, Erlich Y. Genome-wide profiling of heritable and de novo STR variations. Nat Methods. 2017;14(6):590–2.
pubmed: 28436466
pmcid: 5482724
doi: 10.1038/nmeth.4267
Gymrek M, Golan D, Rosset S, Erlich Y. lobSTR: A short tandem repeat profiler for personal genomes. Genome Res. 2012;22(6):1154–62.
pubmed: 22522390
pmcid: 3371701
doi: 10.1101/gr.135780.111
Hall CL, Kesharwani RK, Phillips NR, Planz JV, Sedlazeck FJ, Zascavage RR. Accurate profiling of forensic autosomal STRs using the Oxford Nanopore Technologies MinION device. Forensic Sci Int Genet. 2022;56: 102629.
pubmed: 34837788
doi: 10.1016/j.fsigen.2021.102629
Hall CL, Zascavage RR, Sedlazeck FJ, Planz JV. Potential applications of nanopore sequencing for forensic analysis. Forensic Sci Rev. 2020;32(1):23–54.
pubmed: 32007927
Zascavage RR, Shewale SJ, Planz JV. Deep-sequencing technologies and potential applications in Forensic DNA testing. Forensic Sci Rev. 2013;25(1–2):79–105.
pubmed: 26226852
Pomerantz A, Peñafiel N, Arteaga A, Bustamante L, Pichardo F, Coloma LA, Barrio-Amorós CL, Salazar-Valenzuela D, Prost S. Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building. Gigascience. 2018;7(4):giy033.
pubmed: 29617771
doi: 10.1093/gigascience/giy033
Fernandez F. The greatest impediment to the study of biodiversity in Colombia. Caldasia. 2011;33:iii–v.
Gilbert N. Biodiversity law could stymie research. Nature. 2010;463(7281):598.
pubmed: 20130622
doi: 10.1038/463598a
Blanco MB, Greene LK, Rasambainarivo F, Toomey E, Williams RC, Andrianandrasana L, Larsen PA, Yoder AD. Next-generation technologies applied to age-old challenges in Madagascar. Conserv Genet. 2020;21:785–93.
doi: 10.1007/s10592-020-01296-0
Chang JJ, Ip YC, Ng CS, Huang D. Takeaways from Mobile DNA Barcoding with BentoLab and MinION. Genes (Basel). 2020;11(10):1121.
pubmed: 32987804
doi: 10.3390/genes11101121
Menegon M, Cantaloni C, Rodriguez-Prieto A, Centomo C, Abdelfattah A, Rossato M, Bernardi M, Xumerle L, Loader S, Delledonne M. On site DNA barcoding by nanopore sequencing. PLoS ONE. 2017;12(10): e0184741.
pubmed: 28977016
doi: 10.1371/journal.pone.0184741
Pomerantz A, Sahlin K, Vasiljevic N, Seah A, Lim M, Humble E, Kennedy S, Krehenwinkel H, Winter S, Ogden R, et al. Rapid in situ identification of biological specimens via DNA amplicon sequencing using miniaturized laboratory equipment. Nat Protoc. 2022;17:1415–43.
pubmed: 35411044
doi: 10.1038/s41596-022-00682-x
Asogawa M, Ohno A, Nakagawa S, Ochiai E, Katahira Y, Sudo M, Osawa M, Sugisawa M, Imanishi T. Human short tandem repeat identification using a nanopore-based DNA sequencer: a pilot study. J Hum Genet. 2020;65(1):21–4.
pubmed: 31649301
doi: 10.1038/s10038-019-0688-z
Cornelis S, Gansemans Y, Deleye L, Deforce D, Van Nieuwerburgh F. Forensic SNP genotyping using nanopore MinION sequencing. Sci Rep. 2017;7:41759.
pubmed: 28155888
pmcid: 5290523
doi: 10.1038/srep41759
Ren ZL, Zhang JR, Zhang XM, Liu X, Lin YF, Bai H, Wang MC, Cheng F, Liu JD, Li P, et al. Forensic nanopore sequencing of STRs and SNPs using Verogen’s ForenSeq DNA Signature Prep Kit and MinION. Int J Legal Med. 2021;135(5):1685–93.
pubmed: 33950286
pmcid: 8098014
doi: 10.1007/s00414-021-02604-0
Tytgat O, Gansemans Y, Weymaere J, Rubben K, Deforce D, Van Nieuwerburgh F. Nanopore sequencing of a forensic STR multiplex reveals loci suitable for single-contributor STR profiling. Genes (Basel). 2020;11(4):381.
pubmed: 32244632
doi: 10.3390/genes11040381
Pawar H, Rymbekova A, Cuadros-Espinoza S, Huang X, De Manuel M, Van der Valk T, Lobon I, Alvarez-Estape M, Haber M, Dolgova O, et al. Ghost admixture in eastern gorillas. Nat Ecol Evol. 2023;7:1503–14.
pubmed: 37500909
doi: 10.1038/s41559-023-02145-2
Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20.
pubmed: 24695404
pmcid: 4103590
doi: 10.1093/bioinformatics/btu170
Hoogenboom J, van der Gaag KJ, de Leeuw RH, Sijen T, de Knijff P, Laros JF. FDSTools: A software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise. Forensic Sci Int Genet. 2017;27:27–40.
pubmed: 27914278
doi: 10.1016/j.fsigen.2016.11.007
Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. Integrative genomics viewer. Nat Biotechnol. 2011;29(1):24–6.
pubmed: 21221095
doi: 10.1038/nbt.1754
Huszar TI, Jobling MA, Wetton JH. A phylogenetic framework facilitates Y-STR variant discovery and classification via massively parallel sequencing. Forensic Sci Int Genet. 2018;35:97–106.
pubmed: 29679929
doi: 10.1016/j.fsigen.2018.03.012
Blanchette M, Kent WJ, Riemer C, Elnitski L, Smit AF, Roskin KM, Baertsch R, Rosenbloom K, Clawson H, Green ED, et al. Aligning multiple genomic sequences with the threaded blockset aligner. Genome Res. 2004;14(4):708–15.
pubmed: 15060014
doi: 10.1101/gr.1933104
Sievers F, Higgins DG. Clustal Omega, Accurate Alignment of Very Large Numbers of Sequences. In: Multiple Sequence Alignment Methods. Edited by Russell DJ. Totowa, NJ: Humana Press; 2014:105–116.
Gouy A, Zieger M. STRAF—A convenient online tool for STR data evaluation in forensic genetics. Forensic Sci Int Genet. 2017;30:148–51.
pubmed: 28743032
doi: 10.1016/j.fsigen.2017.07.007
Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics. 2000;155:945–59.
pubmed: 10835412
doi: 10.1093/genetics/155.2.945
Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol. 2005;14(8):2611–20.
pubmed: 15969739
doi: 10.1111/j.1365-294X.2005.02553.x
Earl DA, Volholdt BM. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour. 2012;4:359–61.
doi: 10.1007/s12686-011-9548-7
Jombart T, Ahmed I. Adegenet 1.3–1: new tools for the analysis of genome-wide SNP data. Bioinformatics. 2011;27(21):3070–1.
pubmed: 21926124
pmcid: 3198581
doi: 10.1093/bioinformatics/btr521
R Core Team: R: A language and environment for statistical computing; http://www.R-project.org/ . In. Vienna, Austria: R Foundation for Statistical Computing; 2014.