Maternal peri-conceptional undernourishment perturbs offspring sperm methylome

in Reproduction
Authors:
Paola Toschi Laboratory of Experimental Embryology, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy

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Emanuele Capra Department of Animal Science, Food and Technology – DIANA, and Nutrigenomics and Proteomics Research Center – PRONUTRIGEN, Università Cattolica del Sacro Cuore, Piacenza, Italy
Institute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Lodi, Italy

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Debora A Anzalone Laboratory of Experimental Embryology, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy

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Barbara Lazzari Department of Animal Science, Food and Technology – DIANA, and Nutrigenomics and Proteomics Research Center – PRONUTRIGEN, Università Cattolica del Sacro Cuore, Piacenza, Italy
Institute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Lodi, Italy

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Federica Turri Institute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Lodi, Italy

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Flavia Pizzi Institute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Lodi, Italy

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Pier A Scapolo Laboratory of Experimental Embryology, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy

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Alessandra Stella Institute of Agricultural Biology and Biotechnology (IBBA), National Research Council (CNR), Lodi, Italy

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John L Williams Davies Research Centre, School of Animal and Veterinary Sciences, University of Adelaide, Adelaide, South Australia, Australia

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Paolo Ajmone Marsan Department of Animal Science, Food and Technology – DIANA, and Nutrigenomics and Proteomics Research Center – PRONUTRIGEN, Università Cattolica del Sacro Cuore, Piacenza, Italy

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Pasqualino Loi Laboratory of Experimental Embryology, Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy

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Correspondence should be addressed to P Toschi; Email: ptoschi@unite.it

*(P Toschi and E Capra contributed equally to this work)

DOI:
https://doi.org/10.1530/REP-19-0549
Volume/Issue:
Volume 159: Issue 5
Page Range:
513–523
Article Type:
Research Article
Online Publication Date:
May 2020
Copyright:
© 2020 Society for Reproduction and Fertility 2020
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The genotype of an organism is stable throughout its life; however, its epigenome is dynamic and can be altered in response to environmental factors, such as diet. Inheritance of acquired epigenetic modifications by the next generation occurs through the germline, although the precise mechanisms remain to be elucidated. Here, we used a sheep model to evaluate if modification of the maternal diet (CTR; control, UND: undernutrition; FA: undernutrition and folic acid supplementation) during the peri-conceptional period affects the genome-wide methylation status of the gametes of male offspring. Sperm DNA methylation, measured by Reduced Representation Bisulfite Sequencing (RRBS), identified Differentially Methylated Regions (DMR) in offspring that experienced in utero undernutrition, both in UND (244) and FA (240), compared with CTR. Gene ontology (GO) analysis identified DMRs in categories related to sperm function, therefore we investigated whether the fertilizing capacity of the semen from the three groups differed in an in vitro fertilization assay. Spermatozoa from the undernourished groups showed lower motility and sperm chromatin structure abnormalities, represented by a higher percentage of DNA fragmentation and an increased number of immature cells, compared with CTR. While good quality blastocysts were obtained from all three groups, the proportion of embryos reaching the blastocyst stage was reduced in the UND vs CTR, an effect partially rescued by the FA treatment. The data reported here show that nutritional stress during early pregnancy leads to epigenetic modifications in the semen of the resulting offspring, the effects of which in next generation remain to be elucidated.

Supplementary Materials

    • Supplementary file 1: Sequencing and methylation call statistics. For each sample (control (CTR1, 2, 3), utero undernourished (UND1, 2, 3) and utero undernourished with folic acid supplementation (FA1, 2, 3)), the sequence pairs analysed in total, the total number of cytosines analysed (Tot C), the percentage of cytosine methylated in CpG (mC in CpG), in CHG (mC in CHG), in CHH (mc in CHH) or other (CN) context are reported. H stands for A, T or C whereas N for any nucleotide.
    • Supplementary file 2: Venn diagrams representing the intersections between Highly Methylated Regions (HMRs) in control (CTR), undernutrition (UND) and undernutrition and Folic Acid supplementation (FA) animals in the three comparisons (CTR vs UND, CTR vs FA, UND vs FA) for reduced representation bisulfite sequencing (RRBS) and methylation enrichment and sequencing (Methyl-Seq) datasets. For each dataset, the intersect between 25th percentile of the HMRs was determined and calculated as a percentage.
    • Supplementary file 3: List of differentially methylated regions (DMRs) and DMR associated genes founded in the comparison between different experimental groups (CTR, UND, FA). We identified 244 DMRs in CTR vs UND, 240 DMRs in CTR vs FA and 106 DMRs in UND vs FA. Annotation of the 244, 240 and 106 DMRs identified 75, 69 and 38 DMR associated genes.

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Print ISSN:
1470-1626
Online ISSN:
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  • Alsheimer M, Liebe B, Sewell L, Stewart CL, Scherthan H & Benavente R 2004 Disruption of spermatogenesis in mice lacking A-type lamins. Journal of Cell Science 117 1173–1178. (https://doi.org/10.1242/jcs.00975)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Anderson OS, Sant KE & Dolinoy DC 2012 Nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and DNA methylation. Journal of Nutritional Biochemistry 23 853–859. (https://doi.org/10.1016/j.jnutbio.2012.03.003)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Anzalone DA, Iuso D, Czernik M, Ptak G & Loi P 2016 Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development. Journal of Assisted Reproduction and Genetics 33 757–763. (https://doi.org/10.1007/s10815-016-0709-1)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Ashe A, Sapetschnig A, Weick EM, Mitchell J, Bagijn MP, Cording AC, Doebley AL, Goldstein LD, Lehrbach NJ & Le Pen J et al. 2012 piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans. Cell 150 88–99. (https://doi.org/10.1016/j.cell.2012.06.018)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA & Robinson JS 1993 Fetal nutrition and cardiovascular disease in adult life. Lancet 341 938–941. (https://doi.org/10.1016/0140-6736(93)91224-a)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Bernal AJ & Jirtle RL 2010 Epigenomic disruption: the effects of early developmental exposures. Birth Defects Research: Part A, Clinical and Molecular Teratology 88 938–944. (https://doi.org/10.1002/bdra.20685)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman WH, Pagès F, Trajanoski Z & Galon J 2009 ClueGO: a cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25 1091–1093. (https://doi.org/10.1093/bioinformatics/btp101)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Borowska A, Szwaczkowski T, Kamiński S, Hering DM, Kordan W & Lecewicz M 2018 Identification of genome regions determining semen quality in Holstein-Friesian bulls using information theory. Animal Reproduction Science 192 206–215. (https://doi.org/10.1016/j.anireprosci.2018.03.012)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Butler H & Juurlink BHJ 1987 An Atlas for Staging Mammalian and Chick Embryos, p. 218. Boca Raton Fla: CRC Press, Taylor & Francis Group.

  • Capra E, Turri F, Lazzari B, Cremonesi P, Gliozzi TM, Fojadelli I, Stella A & Pizzi F 2017 Small RNA sequencing of cryopreserved semen from single bull revealed altered miRNAs and piRNAs expression between high- and low-motile sperm populations. BMC Genomics 18 14. (https://doi.org/10.1186/s12864-016-3394-7)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Cetin I, Berti C & Calabrese S 2010 Role of micronutrients in the periconceptional period. Human Reproduction Update 16 80–95. (https://doi.org/10.1093/humupd/dmp025)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Daxinger L & Whitelaw E 2012 Understanding transgenerational epigenetic inheritance via the gametes in mammals. Nature Reviews: Genetics 13 153–162. (https://doi.org/10.1038/nrg3188)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Evenson D & Jost L 2000 Sperm chromatin structure assay is useful for fertility assessment. Methods in Cell Science 22 169–189. (https://doi.org/10.1023/a:1009844109023)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Evenson DP, Darzynkiewicz Z & Melamed MR 1980 Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210 1131–1133. (https://doi.org/10.1126/science.7444440)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P & Claussen OP 1999 Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Human Reproduction 14 1039–1049. (https://doi.org/10.1093/humrep/14.4.1039)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Everitt GC 1964 Maternal undernutrition and retarded fetal development in merino sheep. Nature 201 1341–1342. (https://doi.org/10.1038/2011341a0)

  • Faulk C & Dolinoy DC 2013 Complex phenotypes: epigenetic manifestation of environmental exposures. In Environmental Epigenomics in Health and Disease. Eds Jirtle RL & Tyson FL. Springer.

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Ford SP, Hess BW, Schwope MM, Nijland MJ, Gilbert JS, Vonnahme KA, Means WJ, Han H & Nathanielsz PW 2007 Maternal undernutrition during early to mid-gestation in the ewe results in altered growth, adiposity, and glucose tolerance in male offspring. Journal of Animal Science 85 1285–1294.(https://doi.org/10.2527/jas.2005-624)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Gliozzi TM, Turri F, Manes S, Cassinelli C & Pizzi F 2017 The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen. Animal 11 1975–1982. (https://doi.org/10.1017/S1751731117000684)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Gluckman PD, Hanson MA & Beedle AS 2007 Non-genomic transgenerational inheritance of disease risk. BioEssays 29 145–154. (https://doi.org/10.1002/bies.20522)

  • Greer EL, Maures TJ, Hauswirth AG, Green EM, Leeman DS, Maro GS, Han S, Banko MR, Gozani O & Brunet A 2010 Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans. Nature 466 383–387. (https://doi.org/10.1038/nature09195)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Hackett JA & Surani MA 2013 DNA methylation dynamics during the mammalian life cycle. Philosophical Transactions of the Royal Society of London: Series B, Biological Sciences 368 20110328. (https://doi.org/10.1098/rstb.2011.0328)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Hill PWS, Leitch HG, Requena CE, Sun Z, Amouroux R, Roman-Trufero M, Borkowska M, Terragni J, Vaisvila R & Linnett S et al. 2018 Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte. Nature 555 392–396. (https://doi.org/10.1038/nature25964)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Huypens P, Sass S, Wu M, Dyckhoff D, Tschöp M, Theis F, Marschall S, Hrabě de Angelis M & Beckers J 2016 Epigenetic germline inheritance of diet-induced obesity and insulin resistance. Nature Genetics 48 497–499. (https://doi.org/10.1038/ng.3527)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Januskauska A, Johannisson A & Rodriguez-Martinez H 2001 Assessment of sperm quality through fluorometry and sperm chromatin structure assay in relation to field fertility of frozen-thawed semen from Swedish AI bulls. Theriogenology 55 947–961. (https://doi.org/10.1016/s0093-691x(01)00456-3)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Langley SC & Jackson AA 1994 Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clinical Science 86 217–222; discussion 121. (https://doi.org/10.1042/cs0860217)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Langley-Evans SC, Gardner DS & Welham SJ 1998 Intrauterine programming of cardiovascular disease by maternal nutritional status. Nutrition 14 39–47. (https://doi.org/10.1016/s0899-9007(97)00391-2)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Ledda S, Bogliolo L, Bebbere D, Ariu F & Pirino S 2010 Characterization, isolation and culture of primordial germ cells in domestic animals: recent progress and insights from the ovine species. Theriogenology 74 534–543. (https://doi.org/10.1016/j.theriogenology.2010.05.011)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Martínez D, Pentinat T, Ribó S, Daviaud C, Bloks VW, Cebrià J, Villalmanzo N, Kalko SG, Ramón-Krauel M & Díaz R et al. 2014 In utero undernutrition in male mice programs liver lipid metabolism in the second-generation offspring involving altered LXRa DNA methylation. Cell Metabolism 19 941–951. (https://doi.org/10.1016/j.cmet.2014.03.026)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Messerschmidt DM, Knowles BB & Solter D 2014 DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos. Genes and Development 28 812–828. (https://doi.org/10.1101/gad.234294.113)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Nakagawa T, Zhang T, Kushi R, Nakano S, Endo T, Nakagawa M, Yanagihara N, Zarkower D & Nakayama K 2017 Regulation of mitosis-meiosis transition by the ubiquitin ligase β-TrCP in male germ cells. Development 144 4137–4147. (https://doi.org/10.1242/dev.158485)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Parr RA, Williams AH, Campbell IP, Witcombe GF & Roberts AM 1986 Low nutrition of ewes in early pregnancy and the residual effect on the offspring. Journal of Agricultural Science 106 81–87. (https://doi.org/10.1017/S0021859600061773)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Patti ME 2013 Intergenerational programming of metabolic disease: evidence from human populations and experimental animal models. Cellular and Molecular Life Sciences 70 1597–1608. (https://doi.org/10.1007/s00018-013-1298-0)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Peris SI, Morrier A, Dufour M & Bailey JL 2004 Cryopreservation of ram semen facilitates sperm DNA damage: relationship between sperm andrological parameters and the sperm chromatin structure assay. Journal of Andrology 25 224–233. (https://doi.org/10.1002/j.1939-4640.2004.tb02782.x)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Radford EJ, Ito M, Shi H, Corish JA, Yamazawa K, Isganaitis E, Seisenberger S, Hore TA, Reik W & Erkek S et al. 2014 In utero effects. In utero undernourishment perturbs the adult sperm methylome and intergenerational metabolism. Science 345 1255903. (https://doi.org/10.1126/science.1255903)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Rae MT, Kyle CE, Miller DW, Hammond AJ, Brooks AN & Rhind SM 2002 The effects of undernutrition, in utero, on reproductive function in adult male and female sheep. Animal Reproduction Science 72 63–71. (https://doi.org/10.1016/s0378-4320(02)00068-4)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Rando OJ 2012 Daddy issues: Paternal effects on pheno- type. Cell 151 702–708. ( https://doi.org/10.1016/j.cell.2012.10.020)

  • Ren J, Cheng Y, Ming ZH, Dong XY, Zhou YZ, Ding GL, Pang HY, Rahman TU, Akbar R & Huang HF et al. 2018 Intrauterine hyperglycemia exposure results in intergenerational inheritance via DNA methylation reprogramming on F1 PGCs. Epigenetics and Chromatin 11 20. (https://doi.org/10.1186/s13072-018-0192-2)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Robinson JJ & McDonald I 1979 Ovine prenatal growth, its mathematical description and the effects of maternal nutrition. Annales de Biologie Animale Biochimie Biophysique 19 225–234. (https://doi.org/10.1051/rnd:19790210)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Rodgers AB, Morgan CP, Leu NA & Bale TL 2015 Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress. PNAS 112 13699–13704. (https://doi.org/10.1073/pnas.1508347112)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Rotman T, Etkovitz N, Spiegel A, Rubinstein S & Breitbart H 2010 Protein kinase A and protein kinase C(alpha)/PPP1CC2 play opposing roles in the regulation of phosphatidylinositol 3-kinase activation in bovine sperm. Reproduction 140 43–56. (https://doi.org/10.1530/REP-09-0314)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Sailer BL, Jost LK & Evenson DP 1995 Mammalian sperm DNA susceptibility to in situ denaturation associated with the presence of DNA strand breaks as measured by the terminal deoxynucleotidyl transferase assay. Journal of Andrology 16 80–87. (https://doi.org/10.1002/j.1939-4640.1995.tb01735.x)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Saleh RA, Agarwal A, Nelson DR, Nada EA, El-Tonsy MH, Alvarez JG, Thomas AJ Jr & Sharma RK 2002 Increased sperm nuclear DNA damage in normozoospermic infertile men: a prospective study. Fertility and Sterility 78 313–318. (https://doi.org/10.1016/s0015-0282(02)03219-3)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Schinckel PG & Short BF 1961 Influence of nutritional level during pre-natal and early post-natal life on adult fleece and body characters. Australian Journal of Agricultural Research 12 176–202. (https://doi.org/10.1071/AR9610176)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Seisenberger S, Peat JR, Hore TA, Santos F, Dean W & Reik W 2013 Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers. Philosophical Transactions of the Royal Society of London: Series B, Biological Sciences 368 20110330. (https://doi.org/10.1098/rstb.2011.0330)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Seong KH, Li D, Shimizu H, Nakamura R & Ishii S 2011 Inheritance of stress-induced, ATF-2-dependent epigenetic change. Cell 145 1049–1061. (https://doi.org/10.1016/j.cell.2011.05.029)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Shen J, Chen W, Shao B, Qi Y, Xia Z, Wang F, Wang L, Guo X, Huang X & Sha J 2014 Lamin A/C proteins in the spermatid acroplaxome are essential in mouse spermiogenesis. Reproduction 148 479–487. (https://doi.org/10.1530/REP-14-0012)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Siklenka K, Erkek S, Godmann M, Lambrot R, McGraw S, Lafleur C, Cohen T, Xia J, Suderman M & Hallett M et al. 2015 Disruption of histone methylation in developing sperm impairs offspring health transgenerationally. Science 350 aab2006. (https://doi.org/10.1126/science.aab2006)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Sinclair KD, Karamitri A & Gardner DS 2010 Dietary regulation of developmental programming in ruminants: epigenetic modifications in the germline. Society of Reproduction and Fertility Supplement 67 59–72. (https://doi.org/10.5661/RDR-VII-59)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Sturn A, Quackenbush J & Trajanoski Z 2002 Genesis: cluster analysis of microarray data. Bioinformatics 18 207–208. (https://doi.org/10.1093/bioinformatics/18.1.207)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Trasler JM 2009 Epigenetics in spermatogenesis. Molecular and Cellular Endocrinology 306 33–36. (https://doi.org/10.1016/j.mce.2008.12.018)

  • Van Soom A, Vandaele L, Goossens K, Heras S, Wydooghe E, Rahman MB, Kamal MM, Van Eetvelde M, Opsomer G & Peelman L 2013 Epigenetics and the periconception environment in ruminants. Proceeding of the Belgian Royal Academies of Medicine 2 1–23.

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Wei Y, Schatten H & Sun QY 2015 Environmental epigenetic inheritance through gametes and implications for human reproduction. Human Reproduction Update 21 194–208. (https://doi.org/10.1093/humupd/dmu061)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Xue J, Schoenrock SA, Valdar W, Tarantino LM & Ideraabdullah FY 2016 Maternal vitamin D depletion alters DNA methylation at imprinted loci in multiple generations. Clinical Epigenetics 8 107. (https://doi.org/10.1186/s13148-016-0276-4)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Zambrano E, Rodrigez-González GL, Guzmán C, García-Becerra R, Boeck L, Díaz L, Menjivar M, Larrea F & Nathanielsz PW 2005 A maternal low protein diet during pregnancy and lactation in the rat impairs male reproductive development. Journal of Physiology 563 275–284. (https://doi.org/10.1113/jphysiol.2004.078543)

    • PubMed
    • Search Google Scholar
    • Export Citation

  • Ziller MJ, Hansen KD, Meissner A & Aryee MJ 2015 Coverage recommendations for methylation analysis by whole-genome bisulfite sequencing. Nature Methods 12 230–232. (https://doi.org/10.1038/nmeth.3152)

    • PubMed
    • Search Google Scholar
    • Export Citation