Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.

Environmental Protection College 3320 Velenje Slovenia

Faculty of Agriculture University of Zagreb 10000 Zagreb Croatia

Faculty of Mathematics Natural Sciences and Information Technologies University of Primorska 6000 Koper Slovenia

Institute of Vertebrate Biology of the Czech Academy of Sciences 60365 Brno Czech Republic

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Leroy G., Carroll E.L., Bruford M.W., DeWoody J.A., Strand A., Waits L., Wang J. Next-generation metrics for monitoring genetic erosion within populations of conservation concern. Evol. Appl. 2018;11:1066–1083. doi: 10.1111/eva.12564. PubMed DOI PMC

Eizaguirre C., Baltazar-Soares M. Evolutionary conservation-evaluating the adaptive potential of species. Evol. Appl. 2014;7:963–967. doi: 10.1111/eva.12227. DOI

Funk W.C., McKay J.K., Hohenlohe P.A., Allendorf F.W. Harnessing genomics for delineating conservation units. Trends Ecol. Evol. 2012;27:489–496. doi: 10.1016/j.tree.2012.05.012. PubMed DOI PMC

Sommer S. The importance of immune gene variability (MHC) in evolutionary ecology and conservation. Front. Zool. 2005;2:16. doi: 10.1186/1742-9994-2-16. PubMed DOI PMC

Moreno-Santillán D.D., Lacey E.A., Gendron D., Ortega J. Genetic variation at exon 2 of the MHC class II DQB locus in blue whale (Balaenoptera musculus) from the Gulf of California. PLoS ONE. 2016;11:1–15. doi: 10.1371/journal.pone.0141296. PubMed DOI PMC

Spurgin L.G., Richardson D.S. How pathogens drive genetic diversity: MHC, mechanisms and misunderstandings. Proc. R. Soc. B Biol. Sci. 2010;277:979–988. doi: 10.1098/rspb.2009.2084. PubMed DOI PMC

O’Connor E.A., Strandh M., Hasselquist D., Nilsson J.Å., Westerdahl H. The evolution of highly variable immunity genes across a passerine bird radiation. Mol. Ecol. 2016;25:977–989. doi: 10.1111/mec.13530. PubMed DOI

Reuter J.A., Spacek D.V., Snyder M.P. High-Throughput Sequencing Technologies. Mol. Cell. 2015;58:586–597. doi: 10.1016/j.molcel.2015.05.004. PubMed DOI PMC

Babik W., Taberlet P., Ejsmond M.J., Radwan J. New generation sequencers as a tool for genotyping of highly polymorphic multilocus MHC system. Mol. Ecol. Resour. 2009;9:713–719. doi: 10.1111/j.1755-0998.2009.02622.x. PubMed DOI

Zagalska-Neubauer M., Babik W., Stuglik M., Gustafsson L., Cichoń M., Radwan J. 454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher. BMC Evol. Biol. 2010;10:395. doi: 10.1186/1471-2148-10-395. PubMed DOI PMC

Lighten J., van Oosterhout C., Paterson I.G., McMullan M., Bentzen P. Ultra-deep Illumina sequencing accurately identifies MHC class IIb alleles and provides evidence for copy number variation in the guppy (Poecilia reticulata) Mol. Ecol. Resour. 2014;14:753–767. doi: 10.1111/1755-0998.12225. PubMed DOI

Grogan K.E., McGinnis G.J., Sauther M.L., Cuozzo F.P., Drea C.M. Next-generation genotyping of hypervariable loci in many individuals of a non-model species: Technical and theoretical implications. BMC Genomics. 2016;17:204. doi: 10.1186/s12864-016-2503-y. PubMed DOI PMC

Huang K., Zhang P., Dunn D.W., Wang T., Mi R., Li B. Assigning alleles to different loci in amplifications of duplicated loci. Mol. Ecol. Resour. 2019;19:1240–1253. doi: 10.1111/1755-0998.13036. PubMed DOI

Lighten J., van Oosterhout C., Bentzen P. Critical review of NGS analyses for de novo genotyping multigene families. Mol. Ecol. 2014;23:3957–3972. doi: 10.1111/mec.12843. PubMed DOI

Biedrzycka A., Sebastian A., Migalska M., Westerdahl H., Radwan J. Testing genotyping strategies for ultra-deep sequencing of a co-amplifying gene family: MHC class I in a passerine bird. Mol. Ecol. Resour. 2017;17:642–655. doi: 10.1111/1755-0998.12612. PubMed DOI

Rekdal S.L., Anmarkrud J.A., Johnsen A., Lifjeld J.T. Genotyping strategy matters when analyzing hypervariable major histocompatibility complex-Experience from a passerine bird. Ecol. Evol. 2018;8:1680–1692. doi: 10.1002/ece3.3757. PubMed DOI PMC

Babik W. Methods for MHC genotyping in non-model vertebrates. Mol. Ecol. Resour. 2010;10:237–251. doi: 10.1111/j.1755-0998.2009.02788.x. PubMed DOI

Sunnucks P., Wilson A.C.C., Beheregaray L.B., Zenger K., French J., Taylor A.C. SSCP is not so difficult: The application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology. Mol. Ecol. 2000;9:1699–1710. doi: 10.1046/j.1365-294x.2000.01084.x. PubMed DOI

Garrigan D., Hedrick P.W. Class I MHC polymorphism and evolution in endangered California Chinook and other Pacific salmon. Immunogenetics. 2001;53:483–489. doi: 10.1007/s002510100352. PubMed DOI

Noakes M.A., Reimer T., Phillips R.B. Genotypic characterization of an MHC class II locus in lake trout (Salvelinus namaycush) from Lake Superior by single-stranded conformational polymorphism analysis and reference strand-mediated conformational analysis. Mar. Biotechnol. 2003;5:270–278. doi: 10.1007/s10126-002-0079-9. PubMed DOI

Hughes A.L., Yeager M. Natural selection at major histocompatibility complex loci of vertebrates. Annu. Rev. Genet. 1998;32:415–435. doi: 10.1146/annurev.genet.32.1.415. PubMed DOI

Schaschl H., Goodman S.J., Suchentrunk F. Sequence analysis of the MHC class II DRB alleles in Alpine chamois (Rupicapra r. rupicapra) Dev. Comp. Immunol. 2004;28:265–277. doi: 10.1016/j.dci.2003.08.003. PubMed DOI

Číková D., de Bellocq J.G., Baird S.J.E., Piálek J., Bryja J. Genetic structure and contrasting selection pattern at two major histocompatibility complex genes in wild house mouse populations. Heredity. 2011;106:727–740. doi: 10.1038/hdy.2010.112. PubMed DOI PMC

Bryja J., Galan M., Charbonnel N., Cosson J.F. Analysis of major histocompatibility complex class II gene in water voles using capillary electrophoresis-single stranded conformation polymorphism. Mol. Ecol. Notes. 2005;5:173–176. doi: 10.1111/j.1471-8286.2004.00855.x. DOI

Kumar S., Stecher G., Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol. Evol. 2016;33:1870–1874. doi: 10.1093/molbev/msw054. PubMed DOI PMC

Sebastian A., Herdegen M., Migalska M., Radwan J. Amplisas: A web server for multilocus genotyping using next-generation amplicon sequencing data. Mol. Ecol. Resour. 2016;16:498–510. doi: 10.1111/1755-0998.12453. PubMed DOI

Fuselli S., Baptista R.P., Panziera A., Magi A., Guglielmi S., Tonin R., Benazzo A., Bauzer L.G., Mazzoni C.J., Bertorelle G. A new hybrid approach for MHC genotyping: High-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra) Heredity. 2018;121:293–303. doi: 10.1038/s41437-018-0070-5. PubMed DOI PMC

Zemanová B., Hájková P., Hájek B., Martínková N., Mikulíček P., Zima J., Bryja J. Extremely low genetic variation in endangered Tatra chamois and evidence for hybridization with an introduced Alpine population. Conserv. Genet. 2015;16:729–741. doi: 10.1007/s10592-015-0696-2. DOI

Mona S., Crestanello B., Bankhead-Dronnet S., Pecchioli E., Ingrosso S., D’Amelio S., Rossi L., Meneguz P.G., Bertorelle G. Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois. Mol. Ecol. 2008;17:4053–4067. doi: 10.1111/j.1365-294X.2008.03892.x. PubMed DOI

Leigh J.W., Bryant D. POPART: Full-feature software for haplotype network construction. Methods Ecol. Evol. 2015;6:1110–1116. doi: 10.1111/2041-210X.12410. DOI

Schaschl H., Wandeler P., Suchentrunk F., Obexer-Ruff G., Goodman S.J. Selection and recombination drive the evolution of MHC class II DRB diversity in ungulates. Heredity. 2006;97:427–437. doi: 10.1038/sj.hdy.6800892. PubMed DOI

Alvarez-Busto J., García-Etxebarria K., Herrero J., Garin I., Jugo B.M. Diversity and evolution of the Mhc-DRB1 gene in the two endemic Iberian subspecies of Pyrenean chamois, Rupicapra pyrenaica. Heredity. 2007;99:406–413. doi: 10.1038/sj.hdy.6801016. PubMed DOI

Sommer S., Courtiol A., Mazzoni C.J. MHC genotyping of non-model organisms using next-generation sequencing: A new methodology to deal with artefacts and allelic dropout. BMC Genomics. 2013;14:542. doi: 10.1186/1471-2164-14-542. PubMed DOI PMC

Montero B.K., Refaly E., Ramanamanjato J.B., Randriatafika F., Rakotondranary S.J., Wilhelm K., Ganzhorn J.U., Sommer S. Challenges of next-generation sequencing in conservation management: Insights from long-term monitoring of corridor effects on the genetic diversity of mouse lemurs in a fragmented landscape. Evol. Appl. 2019;12:425–442. doi: 10.1111/eva.12723. PubMed DOI PMC

Radwan J., Zagalska-Neubauer M., Cichoń M., Sendecka J., Kulma K., Gustafsson L., Babik W. MHC diversity, malaria and lifetime reproductive success in collared flycatchers. Mol. Ecol. 2012;21:2469–2479. doi: 10.1111/j.1365-294X.2012.05547.x. PubMed DOI