Genetic diversity and structure in wild Robusta coffee (Coffea canephora A. Froehner) populations in Yangambi (DR Congo) and their relation to forest disturbance.
Depecker, Jonas; Verleysen, Lauren; Asimonyio, Justin Aet al.
[en] Degradation and regeneration of tropical forests can strongly affect gene flow in understorey species, resulting in genetic erosion and changes in genetic structure. Yet, these processes remain poorly studied in tropical Africa. Coffea canephora is an economically important species, found in the understorey of tropical rainforests of Central and West Africa, and the genetic diversity harboured in its wild populations is vital for sustainable coffee production worldwide. Here, we aimed to quantify genetic diversity, genetic structure, and pedigree relations in wild C. canephora populations, and we investigated associations between these descriptors and forest disturbance and regeneration. Therefore, we sampled 256 C. canephora individuals within 24 plots across three forest categories in Yangambi (DR Congo), and used genotyping-by-sequencing to identify 18,894 SNPs. Overall, we found high genetic diversity, and no evidence of genetic erosion in C. canephora in disturbed old-growth forest, as compared to undisturbed old-growth forest. In addition, an overall heterozygosity excess was found in all populations, which was expected for a self-incompatible species. Genetic structure was mainly a result of isolation-by-distance, reflecting geographical location, with low to moderate relatedness at finer scales. Populations in regrowth forest had lower allelic richness than populations in old-growth forest and were characterised by a lower inter-individual relatedness and a lack of isolation-by-distance, suggesting that they originated from different neighbouring populations and were subject to founder effects. Wild Robusta coffee populations in the study area still harbour high levels of genetic diversity, yet careful monitoring of their response to ongoing forest degradation remains required.
Disciplines :
Agriculture & agronomy
Author, co-author :
Depecker, Jonas ; Division of Ecology, Evolution and Biodiversity Conservation, KU Leuven, Leuven, Belgium. Jonas.depecker@kuleuven.be ; Meise Botanic Garden, Meise, Belgium. Jonas.depecker@kuleuven.be ; KU Leuven Plant Institute, Leuven, Belgium. Jonas.depecker@kuleuven.be
Verleysen, Lauren ; Division of Ecology, Evolution and Biodiversity Conservation, KU Leuven, Leuven, Belgium. Lauren.verleysen@ilvo.vlaanderen.be ; Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food, Melle, Belgium. Lauren.verleysen@ilvo.vlaanderen.be
Asimonyio, Justin A; Centre de Surveillance de la Biodiversité et Université de Kisangani, Kisangani, Democratic Republic of the Congo
Hatangi, Yves; Meise Botanic Garden, Meise, Belgium ; Université de Kisangani, Kisangani, Democratic Republic of the Congo
Kambale, Jean-Léon; Centre de Surveillance de la Biodiversité et Université de Kisangani, Kisangani, Democratic Republic of the Congo
Mwanga Mwanga, Ithe; Centre de Recherche en Science Naturelles, Lwiro, Democratic Republic of the Congo
Ebele, Tshimi; Institut National des Etudes et Recherches, Agronomique, Democratic Republic of the Congo
Dhed'a, Benoit; Université de Kisangani, Kisangani, Democratic Republic of the Congo
Bawin, Yves ; Division of Ecology, Evolution and Biodiversity Conservation, KU Leuven, Leuven, Belgium ; Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food, Melle, Belgium
Staelens, Ariane; Plant Sciences Unit, Flanders Research Institute for Agriculture, Fisheries and Food, Melle, Belgium
Genetic diversity and structure in wild Robusta coffee (Coffea canephora A. Froehner) populations in Yangambi (DR Congo) and their relation to forest disturbance.
Alternative titles :
[fr] Diversité et structure génétiques dans les populations sauvages des caféiers Robusta (Coffea canephora A. Froehner) à Yangambi (RD Congo) et leur relation avec les perturbations forestières.
FWO - Fonds Wetenschappelijk Onderzoek Vlaanderen [BE] Foundation for the promotion of biodiversity research in Africa
Funding text :
Fonds Wetenschappelijk Onderzoek
Foundation for the promotion of biodiversity research in Africa, together with Yves Hatangi
Foundation for the promotion of biodiversity research in Africa, together with Jonas Depecker
Aguilar R, Cristóbal-Pérez ED, Balvino-Olvera FJ, Aguilar-Aguilar MDJ, Aguirre-Acosta N, Ashworth L et al. (2019) Habitat fragmentation reduces plant progeny quality: a global synthesis. Ecol Lett 22:1163–1173 DOI: 10.1111/ele.13272
Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc
Barlow J, Lennow GD, Ferreira J, Berenguer E, Lees AC, Nally RM et al. (2016) Anthropogenic disturbance in tropical forests can double biodiversity loss from deforestation. Nature 535:144–147 DOI: 10.1038/nature18326
Barret SC, Eckert CG (1990) Current issues in plant reproductive ecology. Isr J Plant Sci 39:5–12
Bawa KS, Bullock SH, Perry DR, Coville RE, Grayum MH (1985) Reproductive biology of tropical lowland rain forest trees II. Pollination systems. Am J Bot 72:346–356 DOI: 10.1002/j.1537-2197.1985.tb05358.x
Bello C, Galetti M, Pizo MA, Magnago LFS, Roch MF, Lima RA, et al. (2015) Defaunation affects carbon storage in tropical forests. Sci Adv 1:e1501105. https://doi.org/10.1126/sciadv.1501105
Blouin MS (2003) DNA-based methods for pedigree reconstruction and kinship analysis in natural populations. Trends Ecol Evol 18:503–511 DOI: 10.1016/S0169-5347(03)00225-8
Born C, Kjellberg F, Chevallier M-H, Vignes H, Dikangadissi J-T, Sanguié J et al. (2008) Colonization processes and the maintenance of genetic diversity: insight from a pioneer rainforest tree, Aucoumea Klaineana. Proc R Soc B 275:2171–2179 DOI: 10.1098/rspb.2008.0446
Braun M, Dantas L, Esposito T, Pedrosa-Harand A (2020) Strong genetic differentiation on a small geographic scale in the Neotropical rainforest understory tree Paypayrola blanchetiana (Violaceae). Tree Genet Genomes. 10.1007/s11295-020-01477-5
Campbell AJ, Carvalheiro LG, Maués MM, Jaffé R, Giannini TC, Freitas MAB et al. (2018) Anthropogenic disturbance of tropical forests threatens pollination services to açai palm in the Amazon river delta. J Appl Ecol 55:1725–1736 DOI: 10.1111/1365-2664.13086
Chang CC, Chow CC, Tellier LC, Vattikuti S, Purcell SM, Lee JJ (2015) Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience https://doi.org/10.1186/s13742-015-0047-8
Chiriboga-Arroyo F, Jansen M, Bardales-Lozano R, Ismail SA, Thomas E, Garcia M et al. (2021) Genetic threats to the Forest Giants of the Amazon: Habitat degradation effects on the socio-economically important Brazil nut tree (Bertholletia excelsa). Plants People Planet 3:194–210 DOI: 10.1002/ppp3.10166
Cramer PJS, Wellman FL (1957) Review of literature of coffee research in Indonesia. SIC Editorial, Inter-American Institute of Agricultural Sciences
Craparo ACW, Van Asten PJ, Läderach P, Jassogne LT, Grab SW (2015) Coffea arabica yields decline in Tanzania due to climate change: Global implications. Agric Meteorol 207:1–10 DOI: 10.1016/j.agrformet.2015.03.005
Cubry P, De Bellis F, Pot D, Musoli P, Leroy P (2013) Global analysis of Coffea canephora Pierre ex Froehner (Rubiaceae) from the Guineo-Congolese region reveals impacts from climatic refuges and migration effects. Genet Resour Crop Evol 60:483–501 DOI: 10.1007/s10722-012-9851-5
Curtis PG, Slay CM, Harris NL, Tyukavina A, Hansen MC (2018) Classifying drivers of global forest loss. Science 361:1108–1111 DOI: 10.1126/science.aau3445
Da Silva JMC, Tabarelli M (2000) Tree species impoverishment and the future flora of the Atlantic forest of northeast Brazil. Nature 404:72–74 DOI: 10.1038/35003563
Danecek P, Auton A, Abecasis G, Albers CA, Banks E, DePristo MA et al. (2011) The variant call format and VCFtools. Bioinformatics 27:2156–2158 DOI: 10.1093/bioinformatics/btr330
Davis AP, Gole TW, Baena S, Moat J (2012) The impact of climate change on indigenous arabica coffee (Coffea arabica): predicting future trends and identifying priorities. PLoS One. 10.1371/journal.pone.0047981
Denoeud F, Carretero-Paulet L, Dereeper A, Droc G, Guyot R, Pietrella M et al. (2014) The coffee genome provides insight into the convergent evolution of caffeine biosynthesis. Science 345:1181–1184 DOI: 10.1126/science.1255274
Depecker J, Asimonyio JA, Miteho R, Hatangi Y, Kambale J-L, Verleysen L, et al. (2022) The association between rainforest disturbance and recovery, tree community composition, and community traits in the Yangambi area in the Democratic Republic of the Congo. J Trop Ecol. 10.1017/S0266467422000347
Dick CW, Etchelecu G, Austerlitz F (2003) Pollen dispersal of tropical trees (Dinizia excelsa: Fabaceae) by native insects and African honeybees in pristine and fragmented Amazonian rainforest. Mol Ecol 12:753–764 DOI: 10.1046/j.1365-294X.2003.01760.x
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bull 19:11–15
Edwards DP, Socolar JB, Mills SC, Burivalova Z, Koh LP, Wilcove DS (2019) Conservation of tropical forests in the Anthropocene. Curr Biol 29:R1008–R1020 DOI: 10.1016/j.cub.2019.08.026
El Mousadik A, Petit RJ (1996) High level of genetic differentiation for allelic richness among populations of the argan tree [Argania spinosa (L.) Skeels] endemic to Morocco. Theor Appl Genet 92:832–839 DOI: 10.1007/BF00221895
Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, Buckler ES et al. (2011) A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS One. 10.1371/journal.pone.0019379
Ernst C, Mayaux P, Verhegghen A, Bodart C, Christophe M, Defourny P (2013) National forest cover change in Congo Basin: deforestation, reforestation, degradation and regeneration for the years 1990, 2000 and 2005. Glob Chang Biol 19:1173–1187 DOI: 10.1111/gcb.12092
Evanno G, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 14:2611–2620 DOI: 10.1111/j.1365-294X.2005.02553.x
FAO, UNEP (2020) The State of the World’s Forests 2020. In Forests, bio-diversity and people. FAO and UNEP
Ferrão RG, da Fonseca AFA, Ferrão MAG, De Mune LH (2019) Conilon Coffee: the Coffea canephora produced in Brazil. Incaper, Vitória-ES, Brasil
Gardner TA, Barlow J, Chazdon R, Ewers RM, Harvey CA, Peres CA et al. (2009) Prospects for tropical forest biodiversity in a human‐modified world. Ecol Lett 12:561–582 DOI: 10.1111/j.1461-0248.2009.01294.x
García-Fernández C, Sánchez JA, Blanco G (2018) SNP-haplotypes: An accurate approach for parentage and relatedness inference in gilthead sea bream (Sparus aurata). Aquaculture 495:582–591 DOI: 10.1016/j.aquaculture.2018.06.019
Gomez C, Dussert S, Hamon P, Hamon S, De Kochko A, Poncert V (2009) Current genetic differentiation of Coffea canephora pierre ex a. Froehn in the guineo-Congolian african zone: Cumulative impact of ancient climatic changes and recent human activities. BMC Evol Biol 9:167 DOI: 10.1186/1471-2148-9-167
Goudet J (2013) hierfstat: estimation and tests of hierarchical F-statistics. R Package version 0:04–10. http://CRAN.R-project.org/package=hierfstat
Hubbell SP, Foster RB (1986) Biology, chance and history and the structure of tropical rain forest tree communities. In: Diamond JM, Case TJ (eds) Community ecology. Harper and Row, New York, NY, p 314–329
ICO (2022) Coffee Market Report: August 2022. Donwloaded from International Coffee Organization https://www.ico.org/documents/cy2021-22/cmr-0822-e.pdf
Ismail SA, Ghazoul J, Ravikanth G, Kushalappa CG, Uma Shaanker R, Kettle CJ (2017) Evaluating realized seed dispersal across fragmented tropical landscapes: A two‐fold approach using parentage analysis and the neighbourhood model. N Phytol 214:1307–1316 DOI: 10.1111/nph.14427
Jombart T (2008) adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24:1403–1405 DOI: 10.1093/bioinformatics/btn129
Jombart T, Collins C (2015) Analysing genome-wide SNP data using adegenet 2.0.0. https://adegenet.r-forge.r-project.org/files/tutorial-genomics.pdf
Jones AG, Small CM, Paczolt KA, Ratterman NL (2010) A practical guide to methods of parentage analysis. Mol Ecol Resour 10:6–30 DOI: 10.1111/j.1755-0998.2009.02778.x
Jones OR, Wang J (2012) A comparison of four methods for detecting weak genetic structures from maker data. Ecol Evol 2:1048–1055 DOI: 10.1002/ece3.237
Kalinowski ST, Wagner AP, Taper ML (2006) ML-Relate: a computer program for maximum likelihood estimation of relatedness and relationship. Mol Ecol Notes 6:576–579 DOI: 10.1111/j.1471-8286.2006.01256.x
Kearsley E, Verbeeck H, Hufkens K, Van, de Perre F, doetterl S, Baert G et al. (2017) Functional community structure of African monodominant Gilbertiodendron dewevrei forest influenced by local environmental filtering. Ecol Evol 7:295–304 DOI: 10.1002/ece3.2589
Kier G, Mutke J, Dinerstein E, Ricketss TH, Küper W, Kreft H et al. (2005) Global patterns of plant diversity and floristic knowledge. J Biogeogr 32:1107–1116 DOI: 10.1111/j.1365-2699.2005.01272.x
Kiwuka C, Goudsmit E, Tournebize R, Oliveir de Aquino S, Douma JC, Bellanger L et al. (2021) Genetic diversity of native and cultivated Ugandan Robusta coffee (Coffea canephora Pierre ex A. Froehner): Climate influences, breeding potential and diversity conservation. PLoS One 16:e0245965 DOI: 10.1371/journal.pone.0245965
Kreft H, Jetz W (2007) Global patterns and determinants of vascular plant diversity. Proc Natl Acad Sci USA 104:5925–5930 DOI: 10.1073/pnas.0608361104
Lachenaud P, Zhang D (2008) Genetic diversity and population structure in wild stands of cacao trees (Theobroma cacao L.) in French Guiana. Ann For Sci. 10.1051/forest:2008011
Lashermes P, Combes MC, Ribas A, Cenci A, Mahé L, Etienne H (2010) Genetic and physical mapping of the SH3 region that confers resistance to leaf rust in coffee tree (Coffea arabica L.). Tree Genet Genomes 6:973–980 DOI: 10.1007/s11295-010-0306-x
Leroy T, Marraccini P, Dufour M, Montagnon C, Lashermes P, Sabau X et al. (2005) Construction and characterization of a Coffea canephora BAC library to study the organization of sucrose biosynthesis genes. Theor Appl Genet 111:1031–1041 DOI: 10.1007/s00122-005-0018-z
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 14:2078–2079 DOI: 10.1093/bioinformatics/btp352
Li YL, Liu JX (2018) StructureSelector: A web‐based software to select and visualize the optimal number of clusters using multiple methods. Mol Ecol Resour 18:176–177 DOI: 10.1111/1755-0998.12719
Makelele IA, Verheyen K, Boeckx P, Ntaboba LC, Bazirake BM, Ewango C et al. (2021) Afrotropical secondary forests exhibit fast diversity and functional recovery, but slow compositional and carbon recovery after shifting cultivation. J Veg Sci 32:1–13 DOI: 10.1111/jvs.13071
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12 DOI: 10.14806/ej.17.1.200
Mateu-Andrés I, De Paco L (2006) Genetic diversity and the reproductive system in related species of Antirrhinum. Ann Bot 98:1053–1060 DOI: 10.1093/aob/mcl186
Mayr E (1954) Change of genetic environment and evolution. In: Huxley A, Hardy AC, Ford EB (eds) Evolution as a process. Allen and Unwin, London, p 157–180
McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A et al. (2010) The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20:1297–1303 DOI: 10.1101/gr.107524.110
Merot-L’anthoene V, Tournebize R, Darracq O, Rattina V, Lepelley M, Bellanger L et al. (2019) Development and evaluation of a genome-wide Coffee 8.5K SNP array and its application for high-density genetic mapping and for investigating the origin of Coffea arabica L. Plant Biotechnol J 17:1418–1430 DOI: 10.1111/pbi.13066
Musoli P, Cubry P, Aluka P, Billot C, Dufour M, De Bellis F et al. (2009) Genetic differentiation of wild and cultivated populations: diversity of Coffea canephora Pierre in Uganda. Genome 52:634–646 DOI: 10.1139/G09-037
Neushulz EL, Mueller T, Schleuning M, Böhning-Gaese K (2016) Pollination and seed dispersal are the most threatened processes of plant regeneration. Sci Rep 6:1–6
Norden N, Chazdon RL, Chao A, Jiang YH, Vilchez-Alvarado B (2009) Resilience of tropical rain forests: tree community reassembly in secondary forests. Ecol Lett 12:385–394 DOI: 10.1111/j.1461-0248.2009.01292.x
Nowak MD, Davis AP, Anthony F, Yoder AD (2011) Expression and trans-specific polymorphism of self-incompatibility RNases in Coffea (Rubiaceae). PLoS One. 10.1371/journal.pone.0021019
Nyakaana S (2007) Microgeographical genetic structure of forest robusta coffee (Coffea canephora, Pierre), in Kibale National Park, Uganda. Afr J Ecol 45:71–75 DOI: 10.1111/j.1365-2028.2007.00741.x
Oberleitner F, Egger C, Oberdorfer S, Dullinger S, Wanek W, Hietz P (2021) Recovery of aboveground biomass, species richness and composition in tropical secondary forests in SW Costa Rica. Ecol Manag 479:118580 DOI: 10.1016/j.foreco.2020.118580
Olsson O, Nuñez-Iturri G, Smith HG, Ottosson U, Effium EO (2019) Competition, seed dispersal and hunting: what drives germination and seedling survival in an Afrotropical forest? AoB Plants https://doi.org/10.1093/aobpla/plz018
Oryem-Origa H (1999) Fruit and seed ecology of wild Robusta coffee (Coffea canephora Froehner) in Kibale National Park. Uganda Afr J Ecol 37:439–448 DOI: 10.1046/j.1365-2028.1999.00200.x
Peakall R, Smouse RPP (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537–2539 DOI: 10.1093/bioinformatics/bts460
Podani J (2000) Introduction to the exploration of multivariate biological data. Backhuys Publishers, Kerkwere
Poland JA, Rife TW (2012) Genotyping-by-sequencing for plant breeding and genetics. Plant Genome. https://doi.org/10.3835/plantgenome2012.05.0005
Poorter L, Craven D, Jakovac CC, van der Sande MT, Amissah L, Bongers F et al. (2021) Multidimensional tropical forest recovery. Science 374:1370–1376 DOI: 10.1126/science.abh3629
Raj A, Stephens M, Pritchard JK (2014) fastSTRUCTURE: variational inference of population structure in large SNP data sets. Genetics 197:573–589 DOI: 10.1534/genetics.114.164350
RStudio Team (2016) RStudio: Integrated Development for R
Sasaki N, Putz FE (2009) Critical need for new definitions of “forest” and “forest degradation” in global climate change agreements. Conserv Lett 2:226–232 DOI: 10.1111/j.1755-263X.2009.00067.x
Sezen UU, Chazdon RL, Holsinger KE (2007) Multigenerational genetic analysis of tropical secondary regeneration in a canopy palm. Ecology 88:3065–3075 DOI: 10.1890/06-1084.1
Schaumont D, Veeckman E, Van der Jeugt F, Haegeman A, van Glabeke S, Bawin Y et al. (2022) Stack Mapping Anchor Points (SMAP): a versatile suite of tools for read-backed haplotyping. Preprint at bioRxiv https://doi.org/10.1101/2022.03.10.483555
Shapiro AC, Grantham HS, Aguilar-Amuchastegui N, Murray NJ, Gond V, Bonfils D, et al. (2021) Forest condition in the Congo Basin for the assessment of ecosystem conservation status. Ecol Indic. 10.1016/j.ecolind.2020.107268
Silva MDC, Várzea V, Guerra-Guimarães L, Azinheira HG, Fernandez D, Petitot AS et al. (2006) Coffee resistance to the main diseases: leaf rust and coffee berry disease. Braz J Plant Physiol 18:119–147 DOI: 10.1590/S1677-04202006000100010
Theim TJ, Shirk RY, Givnish TJ (2014) Spatial genetic structure in four understorey Psychotria species (Rubiaceae) and implications for tropical forest diversity. Am J Bot 101:1189–1199 DOI: 10.3732/ajb.1300460
Torti SD, Coley PD, Kursar TA (2001) Causes and consequences of monodominance in tropical lowland forests. Am Nat 157:141–153 DOI: 10.1086/318629
Tyukavina A, Hansen MC, Potapov P, Parker D, Okpa C, Stehman SV, et al. (2018) Congo Basin forest loss dominated by increasing smallholder clearing. Sci Adv. 10.1126/sciadv.aat2993
Vanden Abeele S, Janssens SB, Asimonyio Anio J, Bawin Y, Depecker J, Kambale B et al. (2021) Genetic diversity of wild and cultivated Coffea canephora in northeastern DR Congo and the implications for conservation. Am J Bot 108:2425–2434 DOI: 10.1002/ajb2.1769
Vandepitte K, Gristina AS, De Hert K, Meekers T, Roldán-Ruiz I, Honnay O (2012) Recolonization after habitat restoration leads to decreased genetic variation in populations of a terrestrial orchid. Mol Ecol 21:4206–4215 DOI: 10.1111/j.1365-294X.2012.05698.x
Van Vliet N, Muhindo J, Kbale Nyumu J, Mushagalusa O, Nasi R (2018) Mammal depletion processes as evidenced from spatially explicit and temporal local ecological knowledge. Trop Conserv Sci 11:1–16
Vekemans X, Hardy OJ (2004) New insights from fine-scale spatial genetic structure analyses in plant populations. Mol Ecol 13:921–935 DOI: 10.1046/j.1365-294X.2004.02076.x
Vranckx G, Jacquemyn H, Muys B, Honnay O (2012) Meta‐analysis of susceptibility of woody plants to loss of genetic diversity through habitat fragmentation. Conserv Biol 26:228–237 DOI: 10.1111/j.1523-1739.2011.01778.x
Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370
Wellman FL (1961) Coffee. Botany, cultivation, and utilization. Leonard Hill, London
Widmer A, Lexer C (2001) Glacial refugia: sanctuaries for allelic richness, but not for gene diversity. Trends Ecol Evol 16:267–269 DOI: 10.1016/S0169-5347(01)02163-2
Wright S (1932) The role of mutation, inbreeding, crossbreeding and selection in evolution. In: Proceedings of the sixth international congress of genetics. pp 356–366.
Zhang J, Kobert K, Flouri T, Stamatakis A (2014) PEAR: a fast and accurate Illumina Paired-End read merger. Bioinformatics 30:614–620 DOI: 10.1093/bioinformatics/btt593
