MHC genotyping of non-model organisms using next-generation sequencing: a new methodology to deal with artefacts and allelic dropout
Tóm tắt
The Major Histocompatibility Complex (MHC) is the most important genetic marker to study patterns of adaptive genetic variation determining pathogen resistance and associated life history decisions. It is used in many different research fields ranging from human medical, molecular evolutionary to functional biodiversity studies. Correct assessment of the individual allelic diversity pattern and the underlying structural sequence variation is the basic requirement to address the functional importance of MHC variability. Next-generation sequencing (NGS) technologies are likely to replace traditional genotyping methods to a great extent in the near future but first empirical studies strongly indicate the need for a rigorous quality control pipeline. Strict approaches for data validation and allele calling to distinguish true alleles from artefacts are required. We developed the analytical methodology and validated a data processing procedure which can be applied to any organism. It allows the separation of true alleles from artefacts and the evaluation of genotyping reliability, which in addition to artefacts considers for the first time the possibility of allelic dropout due to unbalanced amplification efficiencies across alleles. Finally, we developed a method to assess the confidence level per genotype a-posteriori, which helps to decide which alleles and individuals should be included in any further downstream analyses. The latter method could also be used for optimizing experiment designs in the future. Combining our workflow with the study of amplification efficiency offers the chance for researchers to evaluate enormous amounts of NGS-generated data in great detail, improving confidence over the downstream analyses and subsequent applications.
Tài liệu tham khảo
Klein J: Natural history of the major histocompatibility complex. 1986, New York: Wiley
Sommer S: The importance of immune gene variability (MHC) in evolutionary ecology and conservation. Front Zool. 2005, 2: 16-10.1186/1742-9994-2-16.
Piertney SB, Oliver MK: The evolutionary ecology of the major histocompatibility complex. Heredity. 2006, 96: 7-21.
Kelley J, Walter L, Trowsdale J: Comparative genomics of major histocompatibility complex. Immunogenetics. 2005, 56: 683-695. 10.1007/s00251-004-0717-7.
Babik W: Methods for MHC genotyping in non-model vertebrates. Mol Ecol Res. 2010, 10: 237-251. 10.1111/j.1755-0998.2009.02788.x.
Castro-Prieto A, Wachter B, Sommer S: Cheetah paradigm revisited: MHC diversity in the world’s largest free-ranging population. Mol Biol Evol. 2011, 28 (4): 1455-1468. 10.1093/molbev/msq330.
Lenz TL, Eizaguirre C, Becker S, Reusch TBH: RSCA genotyping of MHC for high-throughput evolutionary studies in the model organism three-spined stickleback Gasterosteus aculeatus. BMC Evol Biol. 2009, 9: 57-10.1186/1471-2148-9-57.
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: 395-10.1186/1471-2148-10-395.
Kanagawa T: Bias and artifacts in multitemplate polymerase chain reactions (PCR). J Biosci Bioeng. 2003, 56: 317-323.
Lenz TL, Becker S: Simple approach to reduce PCR artefact formation leads to reliable genotyping of MHC and other highly polymorphic loci-implications for evolutionary analysis. Gene. 2008, 427: 117-123. 10.1016/j.gene.2008.09.013.
Cummings SM, McMullan M, Joyce DA, Van Oosterhout C: Solutions for PCR, cloning and sequencing errors in population genetic analysis. Conserv Genetics. 2010, 11: 1095-1097. 10.1007/s10592-009-9864-6.
Babik W, Taberlet P, Ejsmond MJ, Radwan J: New generation sequencers as a tool for genotyping of highly polymorphic multilocus MHC system. Mol Ecol Res. 2009, 9: 713-719. 10.1111/j.1755-0998.2009.02622.x.
Galan M, Guivier E, Caraux G, Charbonnel N, Cosson JF: A 454 multiplex sequencing method for rapid and reliable genotyping of highly polymorphic genes in large-scale studies. BMC Genomics. 2010, 11: 296-10.1186/1471-2164-11-296.
Kloch A, Babik W, Bajer A, Sinski E, Radwan J: Effects of an MHC-DRB genotype and allele number on the load of gut parasites in the bank vole Myodes glareolus. Mol Ecol. 2010, 19: 255-265.
Promerová M, Babik W, Bryja J, Albrecht T, Stuglik M, Radwan J: Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine-CE-SSCP and 454 pyrosequencing. Mol Ecol Res. 2012, 12: 285-292. 10.1111/j.1755-0998.2011.03082.x.
Oomen RA, Gillett RM, Kyle CJ: Comparison of 454 pyrosequencing methods for characterizing the major histocompatibility complex of nonmodel species and the advantages of ultra deep coverage. Mol Ecol Res. 2013, 13: 103-116. 10.1111/1755-0998.12027.
Sepil I, Moghadam HK, Huchard E, Sheldon BC: Characterization and 454 pyrosequencing of Major Histocompatibility Complex class I genes in the great tit reveal complexity in a passerine system. BMC Evol Biol. 2012, 12: 68-10.1186/1471-2148-12-68.
Strandh M, Westerdahl H, Pontarp M, Canbäck B, Dubois MP, Miquel C, Taberlet P, Bonadonna F: Major histocompatibility complex class II compatibility, but not class I, predicts mate choice in a bird with highly developed olfaction. Proc R Soc Lond B. 2012, 279: 4457-4463. 10.1098/rspb.2012.1562.
Gilles A, Meglecz E, Pech N, Ferreira S, Malausa T, Martin JF: Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing. BMC Genomics. 2011, 12: 245-10.1186/1471-2164-12-245.
Moore MJ, Dhingra A, Soltis PS: Rapid and accurate pyrosequencing of angiosperm plastid genomes. BMC Plant Biol. 2006, 6: 17-10.1186/1471-2229-6-17.
Huse SM, Huber JA, Morrison HG, Sogin ML, Welch DM: Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol. 2007, 8: R143-10.1186/gb-2007-8-7-r143.
Schad J, Dechmann D, Voigt C, Sommer S: Evidence for the ‘good genes’ theory: association of parasitism and reproductive state with immune gene constitution in the bat, Noctilio albiventris. PLoS One. 2012, 7 (5): e37101-10.1371/journal.pone.0037101.
Schad J, Voigt C, Greiner S, Dechmann D, Sommer S: Independent evolution of functional MHC class II DRB genes in new world bat species. Immunogenetics. 2012, 64 (7): 535-547. 10.1007/s00251-012-0609-1.
Püttker T, Bueno AA, Dos Santos De Barros C, Sommer S, Pardini R: Habitat specialization interacts with habitat amount to determine dispersal success of rodents in fragmented landscapes. J Mamm. 2013, 94 (2): XY-
Püttker T, Dos Santos De Barros C, Martins TK, Sommer S, Pardini R: Suitability of distance metrics as indices of home range size in tropical rodent species. J Mamm. 2012, 93 (1): 115-123. 10.1644/10-MAMM-A-364.1.
Püttker T, Bueno AA, Dos Santos De Barros C, Sommer S, Pardini R: Immigration rates in fragmented landscapes-empirical evidence for the importance of habitat amount for species persistence. PLoS One. 2011, 6 (11): e27963-10.1371/journal.pone.0027963.
Püttker T, Meyer-Lucht Y, Sommer S: Effect of fragmentation on parasite burden (Nematodes) of generalist and specialist small mammal species in secondary forest fragments of the coastal Atlantic Rain Forest, Brazil. Ecol Res. 2008, 23: 207-215. 10.1007/s11284-007-0366-z.
Püttker T, Meyer-Lucht Y, Sommer S: Fragmentation effects on population density of three rodent species in secondary Atlantic Rainforest, Brazil. Stud Neotropical Fauna Environ. 2008, 43 (1): 11-18. 10.1080/01650520701553651.
Püttker T, Pardini R, Meyer-Lucht Y, Sommer S: Responses of five small mammal species to micro-scale variations in vegetation structure in secondary Atlantic Forest remnants, Brazil. BMC Ecol. 2008, 8: 9-10.1186/1472-6785-8-9.
Püttker T, Meyer-Lucht Y, Sommer S: Movement distances of five rodent and two marsupial species in forest fragments of the coastal Atlantic Rainforest, Brazil. Ecotropica. 2006, 12 (2): 131-139.
Sommer S, Taubert R, Schmidt A, Axtner J, Lieckfeldt D: Development of polymorphic microsatellite marker for Delomys sublineatus, an endemic rodent of the Brazilian Atlantic rain forest by a time and cost-efficient enrichment protocol using genomic DNAs of unrelated organisms for cross-hybridization: added to permanent genetic resources database (1 April 2010-31 May 2010). Mol Ecol Res 2010. 2010, 10 (6): 1098-1105.
Balkenhol N, Fernandes F, Cornelius C, Pardini R, Sommer S: Landscape-level comparison of genetic diversity and differentiation in a small mammal inhabiting different fragmented landscapes of the Brazilian Atlantic Forest. Conserv Genet. 2013, 14 (2): 355-367. 10.1007/s10592-013-0454-2.
Schwensow N, Axtner J, Sommer S: Are associations of immune gene expression, body condition and parasite burden detectible in nature? A case study in an endemic rodent from the Brazilian Atlantic Forest. Infect Genet Evol. 2011, 11: 23-30. 10.1016/j.meegid.2010.10.017.
Weyrich A, Axtner J, Sommer S: Selection and validation of reference genes for real-time RT-PCR studies in the non-model species Delomys sublineatus, an endemic Brazilian rodent. Biochem Biophys Res Comm. 2010, 392 (2): 145-149. 10.1016/j.bbrc.2009.12.173.
Sikes RS, Gannon WL, and the Animal Care and Use Committee of the American Society of Mammalogists: Guidelines of the American Society of Mammalogists for the use of wild mammals in research. J Mammal. 2011, 92: 235-253. 10.1644/10-MAMM-F-355.1.
Klein J, Bontrop RE, Dawkins RL, Erlich HA, Gyllensten UB, Heise ER, Jones PP, Parham P, Wakeland EK, Watkins DI: Nomenclature for the major histocompatibility complexes of different species: a proposal. Immunogenetics. 1990, 31 (4): 217-219.
Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res. 2004, 32 (5): 1792-1797. 10.1093/nar/gkh340.
Drummond AJ, Ashton B, Buxton S, Cheung M, Cooper A, Heled J, Kearse M, Moir R, Stones-Havas S, Sturrock S, Thierer T, Wilson A: Geneious v5.5. 2011, available from http://www.geneious.com
Stuglik MT, Radwan J, Babik W: jMHC: software assistant for multilocus genotyping of gene families using next-generation amplicon sequencing. Mol Ecol Res. 2011, 11 (4): 739-742. 10.1111/j.1755-0998.2011.02997.x.
Meglécz E, Piry S, Desmarais E, Galan M, Gilles A, Guivier E, Pech N, Martin J-F: SESAME (SEquence Sorter & AMplicon Explorer): genotyping based on high-throughput multiplex amplicon sequencing). Bioinformatics. 2011, 27 (2): 277-278. 10.1093/bioinformatics/btq641.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28: 2731-2739. 10.1093/molbev/msr121.
R Core Team: A language and environment for statistical computing. 2012, R Austria: R Foundation for Statistical Computing Vienna,http://www.R-project.org/, 3-900051-07-0,
Brown JH, Jardetzky TS, Gorga JC, Stern LJ: Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature. 1993, 364: 33-39. 10.1038/364033a0.