Skip to main content
SpringerLink
Log in

Low-pass genome sequencing: a validated method in clinical cytogenetics

  • Original Investigation
  • Published:
Human Genetics Aims and scope Submit manuscript

Abstract

Clinically significant copy-number variants (CNVs) known to cause human diseases are routinely detected by chromosomal microarray analysis (CMA). Recently, genome sequencing (GS) has been introduced for CNV analysis; however, sequencing depth (determined by sequencing read-length and read-amount) is a variable parameter across different laboratories. Variating sequencing depths affect the CNV detection resolution and also make it difficult for cross-laboratory referencing or comparison. In this study, by using data from 50 samples with high read-depth GS (30×) and the reported clinically significant CNVs, we first demonstrated the optimal read-amount and the most cost-effective read-length for CNV analysis to be 15 million reads and single-end 50 bp (equivalent to a read-depth of 0.25-fold), respectively. In addition, we showed that CNVs at mosaic levels as low as 30% are readily detected, furthermore, CNVs larger than 2.5 Mb are also detectable at mosaic levels as low as 20%. Herein, by conducting a retrospective back-to-back comparison study of low-pass GS versus routine CMA for 532 prenatal, miscarriage, and postnatal cases, the overall diagnostic yield was 22.4% (119/532) for CMA and 23.1% (123/532) for low-pass GS. Thus, the overall relative improvement of the diagnostic yield by low-pass GS versus CMA was ~ 3.4% (4/119). Identification of cryptic and clinically significant CNVs among prenatal, miscarriage, and postnatal cases demonstrated that CNV detection at higher resolutions is warranted for clinical diagnosis regardless of referral indications. Overall, our study supports low-pass GS as the first-tier genetic test for molecular cytogenetic testing.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Availability of data and material

The genome sequencing data in this study have been made available on the CNGB Nucleotide Sequence Archive (CNSA: https://db.cngb.org/cnsa) under the accession number CNP0000901. Code availability: All relative programs are available at https://sourceforge.net/projects/increment-ratio-of-coverage-v2/files/Increment_Ratio_of_Coverage_V2.0.tar.gz/download.

References

  • 1000 Genomes Project Consortium (2015) A global reference for human genetic variation. Nature 526:68–74. https://doi.org/10.1038/nature15393

    Article  CAS  Google Scholar 

  • Brown KH, Dobrinski KP, Lee AS, Gokcumen O, Mills RE, Shi X, Chong WW, Chen JY, Yoo P, David S, Peterson SM, Raj T, Choy KW, Stranger BE, Williamson RE, Zon LI, Freeman JL, Lee C (2012) Extensive genetic diversity and substructuring among zebrafish strains revealed through copy number variant analysis. Proc Natl Acad Sci USA 109:529–534. https://doi.org/10.1073/pnas.1112163109

    Article  PubMed  Google Scholar 

  • Chau MHK, Cao Y, Kwok YKY, Chan S, Chan YM, Wang H, Yang Z, Wong HK, Leung TY, Choy KW (2019) Characteristics and mode of inheritance of pathogenic copy number variants in prenatal diagnosis. Am J Obstet Gynecol 221(493):e1–493. https://doi.org/10.1016/j.ajog.2019.06.007

    Article  CAS  Google Scholar 

  • Chen Y, Bartanus J, Liang D, Zhu H, Breman AM, Smith JL, Wang H, Ren Z, Patel A, Stankiewicz P, Cram DS, Cheung SW, Wu L, Yu F (2017) Characterization of chromosomal abnormalities in pregnancy losses reveals critical genes and loci for human early development. Hum Mutat 38:669–677. https://doi.org/10.1002/humu.23207

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng YK, Wong C, Wong HK, Leung KO, Kwok YK, Suen A, Wang CC, Leung TY, Choy KW (2013) The detection of mosaicism by prenatal BoBs. Prenat Diagn 33:42–49. https://doi.org/10.1002/pd.4006

    Article  CAS  PubMed  Google Scholar 

  • Choy KW, Wang H, Shi M, Chen J, Yang Z, Zhang R, Yan H, Wang Y, Chen S, Chau MHK, Cao Y, Chan OYM, Kwok YK, Zhu Y, Chen M, Leung TY, Dong Z (2019) Prenatal diagnosis of fetuses with increased nuchal translucency by genome sequencing analysis. Front Genet 10:761. https://doi.org/10.3389/fgene.2019.00761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • de Pontual L, Mathieu Y, Golzio C, Rio M, Malan V, Boddaert N, Soufflet C, Picard C, Durandy A, Dobbie A, Heron D, Isidor B, Motte J, Newburry-Ecob R, Pasquier L, Tardieu M, Viot G, Jaubert F, Munnich A, Colleaux L, Vekemans M, Etchevers H, Lyonnet S, Amiel J (2009) Mutational, functional, and expression studies of the TCF4 gene in Pitt-Hopkins syndrome. Hum Mutat 30:669–676. https://doi.org/10.1002/humu.20935

    Article  CAS  PubMed  Google Scholar 

  • Dharmadhikari AV, Gambin T, Szafranski P, Cao W, Probst FJ, Jin W, Fang P, Gogolewski K, Gambin A, George-Abraham JK, Golla S, Boidein F, Duban-Bedu B, Delobel B, Andrieux J, Becker K, Holinski-Feder E, Cheung SW, Stankiewicz P (2014) Molecular and clinical analyses of 16q24.1 duplications involving FOXF1 identify an evolutionarily unstable large minisatellite. BMC Med Genet 15:128. https://doi.org/10.1186/s12881-014-0128-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dong Z, Zhang J, Hu P, Chen H, Xu J, Tian Q, Meng L, Ye Y, Wang J, Zhang M, Li Y, Wang H, Yu S, Chen F, Xie J, Jiang H, Wang W, Choy KW, Xu Z (2016) Low-pass whole-genome sequencing in clinical cytogenetics: a validated approach. Genet Med 18:940–948. https://doi.org/10.1038/gim.2015.199

    Article  CAS  PubMed  Google Scholar 

  • Dong Z, Yan J, Xu F, Yuan J, Jiang H, Wang H, Chen H, Zhang L, Ye L, Xu J, Shi Y, Yang Z, Cao Y, Chen L, Li Q, Zhao X, Li J, Chen A, Zhang W, Wong HG, Qin Y, Zhao H, Chen Y, Li P, Ma T, Wang WJ, Kwok YK, Jiang Y, Pursley AN, Chung JPW, Hong Y, Kristiansen K, Yang H, Pina-Aguilar RE, Leung TY, Cheung SW, Morton CC, Choy KW, Chen ZJ (2019a) Genome sequencing explores complexity of chromosomal abnormalities in recurrent miscarriage. Am J Hum Genet 105:1102–1111. https://doi.org/10.1016/j.ajhg.2019.10.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dong Z, Zhao X, Li Q, Yang Z, Xi Y, Alexeev A, Shen H, Wang O, Ruan J, Ren H, Wei H, Qi X, Li J, Zhu X, Zhang Y, Dai P, Kong X, Kirkconnell K, Alferov O, Giles S, Yamtich J, Kermani BG, Dong C, Liu P, Mi Z, Zhang W, Xu X, Drmanac R, Choy KW, Jiang Y (2019b) Development of coupling controlled polymerizations by adapter-ligation in mate-pair sequencing for detection of various genomic variants in one single assay. DNA Res. https://doi.org/10.1093/dnares/dsz011

    Article  PubMed  PubMed Central  Google Scholar 

  • Foyouzi N, Cedars MI, Huddleston HG (2012) Cost-effectiveness of cytogenetic evaluation of products of conception in the patient with a second pregnancy loss. Fertil Steril 98:151–155. https://doi.org/10.1016/j.fertnstert.2012.04.007

    Article  PubMed  Google Scholar 

  • Goodspeed K, Newsom C, Morris MA, Powell C, Evans P, Golla S (2018) Pitt-Hopkins syndrome: a review of current literature, clinical approach, and 23-patient case series. J Child Neurol 33:233–244. https://doi.org/10.1177/0883073817750490

    Article  PubMed  PubMed Central  Google Scholar 

  • Gross AM, Ajay SS, Rajan V, Brown C, Bluske K, Burns NJ, Chawla A, Coffey AJ, Malhotra A, Scocchia A, Thorpe E, Dzidic N, Hovanes K, Sahoo T, Dolzhenko E, Lajoie B, Khouzam A, Chowdhury S, Belmont J, Roller E, Ivakhno S, Tanner S, McEachern J, Hambuch T, Eberle M, Hagelstrom RT, Bentley DR, Perry DL, Taft RJ (2019) Copy-number variants in clinical genome sequencing: deployment and interpretation for rare and undiagnosed disease. Genet Med 21:1121–1130. https://doi.org/10.1038/s41436-018-0295-y

    Article  CAS  PubMed  Google Scholar 

  • Gu S, Szafranski P, Akdemir ZC, Yuan B, Cooper ML, Magrina MA, Bacino CA, Lalani SR, Breman AM, Smith JL, Patel A, Song RH, Bi W, Cheung SW, Carvalho CM, Stankiewicz P, Lupski JR (2016) Mechanisms for complex chromosomal insertions. PLoS Genet 12:e1006446. https://doi.org/10.1371/journal.pgen.1006446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hay SB, Sahoo T, Travis MK, Hovanes K, Dzidic N, Doherty C, Strecker MN (2018) ACOG and SMFM guidelines for prenatal diagnosis: is karyotyping really sufficient? Prenat Diagn 38:184–189. https://doi.org/10.1002/pd.5212

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang J, Poon LC, Akolekar R, Choy KW, Leung TY, Nicolaides KH (2014) Is high fetal nuchal translucency associated with submicroscopic chromosomal abnormalities on array CGH? Ultrasound Obstet Gynecol 43:620–624. https://doi.org/10.1002/uog.13384

    Article  CAS  PubMed  Google Scholar 

  • Iafrate AJ, Feuk L, Rivera MN, Listewnik ML, Donahoe PK, Qi Y, Scherer SW, Lee C (2004) Detection of large-scale variation in the human genome. Nat Genet 36:949–951. https://doi.org/10.1038/ng1416

    Article  CAS  PubMed  Google Scholar 

  • Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754–1760. https://doi.org/10.1093/bioinformatics/btp324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li X, Chen S, Xie W, Vogel I, Choy KW, Chen F, Christensen R, Zhang C, Ge H, Jiang H, Yu C, Huang F, Wang W, Jiang H, Zhang X (2014) PSCC: sensitive and reliable population-scale copy number variation detection method based on low coverage sequencing. PLoS ONE 9:e85096. https://doi.org/10.1371/journal.pone.0085096

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liang D, Peng Y, Lv W, Deng L, Zhang Y, Li H, Yang P, Zhang J, Song Z, Xu G, Cram DS, Wu L (2014) Copy number variation sequencing for comprehensive diagnosis of chromosome disease syndromes. J Mol Diagn 16:519–526. https://doi.org/10.1016/j.jmoldx.2014.05.002

    Article  CAS  PubMed  Google Scholar 

  • Lin SB, Xie YJ, Chen Z, Zhou Y, Wu JZ, Zhang ZQ, Shi SS, Chen BJ, Fang Q (2015) Improved assay performance of single nucleotide polymorphism array over conventional karyotyping in analyzing products of conception. J Chin Med Assoc 78:408–413. https://doi.org/10.1016/j.jcma.2015.03.010

    Article  PubMed  Google Scholar 

  • Markello TC, Carlson-Donohoe H, Sincan M, Adams D, Bodine DM, Farrar JE, Vlachos A, Lipton JM, Auerbach AD, Ostrander EA, Chandrasekharappa SC, Boerkoel CF, Gahl WA (2012) Sensitive quantification of mosaicism using high density SNP arrays and the cumulative distribution function. Mol Genet Metab 105:665–671. https://doi.org/10.1016/j.ymgme.2011.12.015

    Article  CAS  PubMed  Google Scholar 

  • Miller DT, Adam MP, Aradhya S, Biesecker LG, Brothman AR, Carter NP, Church DM, Crolla JA, Eichler EE, Epstein CJ, Faucett WA, Feuk L, Friedman JM, Hamosh A, Jackson L, Kaminsky EB, Kok K, Krantz ID, Kuhn RM, Lee C, Ostell JM, Rosenberg C, Scherer SW, Spinner NB, Stavropoulos DJ, Tepperberg JH, Thorland EC, Vermeesch JR, Waggoner DJ, Watson MS, Martin CL, Ledbetter DH (2010) Consensus statement: chromosomal microarray is a first-tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. Am J Hum Genet 86:749–764. https://doi.org/10.1016/j.ajhg.2010.04.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Motley WW, Palaima P, Yum SW, Gonzalez MA, Tao F, Wanschitz JV, Strickland AV, Loscher WN, De Vriendt E, Koppi S, Medne L, Janecke AR, Jordanova A, Zuchner S, Scherer SS (2016) De novo PMP2 mutations in families with type 1 Charcot-Marie-Tooth disease. Brain 139:1649–1656. https://doi.org/10.1093/brain/aww055

    Article  PubMed  PubMed Central  Google Scholar 

  • Redin C, Brand H, Collins RL, Kammin T, Mitchell E, Hodge JC, Hanscom C, Pillalamarri V, Seabra CM, Abbott MA, Abdul-Rahman OA, Aberg E, Adley R, Alcaraz-Estrada SL, Alkuraya FS, An Y, Anderson MA, Antolik C, Anyane-Yeboa K, Atkin JF, Bartell T, Bernstein JA, Beyer E, Blumenthal I, Bongers EM, Brilstra EH, Brown CW, Bruggenwirth HT, Callewaert B, Chiang C, Corning K, Cox H, Cuppen E, Currall BB, Cushing T, David D, Deardorff MA, Dheedene A, D'Hooghe M, de Vries BB, Earl DL, Ferguson HL, Fisher H, FitzPatrick DR, Gerrol P, Giachino D, Glessner JT, Gliem T, Grady M, Graham BH, Griffis C, Gripp KW, Gropman AL, Hanson-Kahn A, Harris DJ, Hayden MA, Hill R, Hochstenbach R, Hoffman JD, Hopkin RJ, Hubshman MW, Innes AM, Irons M, Irving M, Jacobsen JC, Janssens S, Jewett T, Johnson JP, Jongmans MC, Kahler SG, Koolen DA, Korzelius J, Kroisel PM, Lacassie Y, Lawless W, Lemyre E, Leppig K, Levin AV, Li H, Li H, Liao EC, Lim C, Lose EJ, Lucente D, Macera MJ, Manavalan P, Mandrile G, Marcelis CL, Margolin L, Mason T, Masser-Frye D, McClellan MW, Mendoza CJ, Menten B, Middelkamp S, Mikami LR, Moe E, Mohammed S, Mononen T, Mortenson ME et al (2017) The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies. Nat Genet 49:36–45. https://doi.org/10.1038/ng.3720

    Article  CAS  PubMed  Google Scholar 

  • Riggs ER, Andersen EF, Cherry AM, Kantarci S, Kearney H, Patel A, Raca G, Ritter DI, South ST, Thorland EC, Pineda-Alvarez D, Aradhya S, Martin CL (2019) Technical standards for the interpretation and reporting of constitutional copy-number variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen). Genet Med. https://doi.org/10.1038/s41436-019-0686-8

    Article  PubMed  PubMed Central  Google Scholar 

  • Trost B, Walker S, Wang Z, Thiruvahindrapuram B, MacDonald JR, Sung WWL, Pereira SL, Whitney J, Chan AJS, Pellecchia G, Reuter MS, Lok S, Yuen RKC, Marshall CR, Merico D, Scherer SW (2018) A comprehensive workflow for read depth-based identification of copy-number variation from whole-genome sequence data. Am J Hum Genet 102:142–155. https://doi.org/10.1016/j.ajhg.2017.12.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang J, Chen L, Zhou C, Wang L, Xie H, Xiao Y, Zhu H, Hu T, Zhang Z, Zhu Q, Liu Z, Liu S, Wang H, Xu M, Ren Z, Yu F, Cram DS, Liu H (2018) Prospective chromosome analysis of 3429 amniocentesis samples in China using copy number variation sequencing. Am J Obstet Gynecol 219:287e1–287e18. https://doi.org/10.1016/j.ajog.2018.05.030

    Article  Google Scholar 

  • Wang JC, Radcliff J, Coe SJ, Mahon LW (2019) Effects of platforms, size filter cutoffs, and targeted regions of cytogenomic microarray on detection of copy number variants and uniparental disomy in prenatal diagnosis: results from 5026 pregnancies. Prenat Diagn 39:137–156. https://doi.org/10.1002/pd.5375

    Article  CAS  PubMed  Google Scholar 

  • Wang H, Dong Z, Zhang R, Chau MHK, Yang Z, Tsang KYC, Wong HK, Gui B, Meng Z, Xiao K, Zhu X, Wang Y, Chen S, Leung TY, Cheung SW, Kwok YK, Morton CC, Zhu Y, Choy KW (2020a) Low-pass genome sequencing versus chromosomal microarray analysis: implementation in prenatal diagnosis. Genet Med 22:500–510. https://doi.org/10.1038/s41436-019-0634-7

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Li Y, Chen Y, Zhou R, Sang Z, Meng L, Tan J, Qiao F, Bao Q, Luo D, Peng C, Wang YS, Luo C, Hu P, Xu Z (2020b) Systematic analysis of copy-number variations associated with early pregnancy loss. Ultrasound Obstet Gynecol 55:96–104. https://doi.org/10.1002/uog.20412

    Article  CAS  PubMed  Google Scholar 

  • Zhang L, Bai W, Yuan N, Du Z (2019) Comprehensively benchmarking applications for detecting copy number variation. PLoS Comput Biol 15:e1007069. https://doi.org/10.1371/journal.pcbi.1007069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This project is supported by the National Natural Science Foundation of China (31801042, 81741004 and 81741009), the Health and Medical Research Fund (04152666) and the Health Department of Guangxi Province (Z20190782). Zirui Dong thanks the Hong Kong Obstetrical & Gynaecological Trust Fund for the support.

Author information

Author notes

  1. Matthew Hoi Kin Chau, Huilin Wang and Yunli Lai contributed equally.

Authors and Affiliations

  1. Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China

    Matthew Hoi Kin Chau, Yanyan Zhang, Tak Yeung Leung, Jacqueline Pui Wah Chung, Yvonne K. Kwok, Kwong Wai Choy & Zirui Dong

  2. Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China

    Matthew Hoi Kin Chau, Yanyan Zhang, Zihan Chen, Tak Yeung Leung, Yvonne K. Kwok, Kwong Wai Choy & Zirui Dong

  3. Hong Kong Hub of Paediatric Excellence, The Chinese University of Hong Kong, Hong Kong, China

    Matthew Hoi Kin Chau, Shuk Ching Chong, Kwong Wai Choy & Zirui Dong

  4. Department of Center Laboratory, Maternal-Fetal Medicine Institute, Shenzhen Key Laboratory of Birth Defects Research, Birth Defects Prevention Research and Transformation Team, Baoan Maternity and Child Health Hospital Affiliated to Jinan University School of Medicine, Shenzhen, 518100, China

    Huilin Wang, Yanfang Wang, Yuanfang Zhu & Likuan Xiong

  5. Birth Defects Prevention and Control Institute of Guangxi Zhuang Autonomous Region, Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region, Nanning, 530000, Guangxi, China

    Yunli Lai, Fuben Xu, Yanqing Tang & Weihong Wei

  6. Genetic and Metabolic Central Laboratory, Guangxi Zhuang Autonomous Region Women and Children Care Hospital, Nanning, 530000, Guangxi, China

    Yunli Lai, Fuben Xu, Yanqing Tang & Weihong Wei

  7. The Chinese University of Hong Kong-Baylor College of Medicine Joint Center For Medical Genetics, Hong Kong, China

    Tak Yeung Leung, Shuk Ching Chong & Kwong Wai Choy

  8. Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, China

    Shuk Ching Chong

Authors
  1. Matthew Hoi Kin Chau
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Huilin Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yunli Lai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yanyan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fuben Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yanqing Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yanfang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zihan Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Tak Yeung Leung
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jacqueline Pui Wah Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yvonne K. Kwok
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Shuk Ching Chong
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Kwong Wai Choy
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yuanfang Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Likuan Xiong
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Weihong Wei
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Zirui Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

ZD, MHKC, HW, YL, TYL, YK, LX, YZ, WW, and KWC designed the study. HW, YL, TYL, JPWC, SCC, YZ, LX and WW collected the samples and followed up. MHKC, HW, YL, FX, YT, YW and ZC performed CMA and low-pass GS. ZD, MHKC and YK performed the analysis and data interpretation. MHKC, HW and YL conducted the validation. ZD, MHKC, HW, YL, and KWC wrote the manuscript.

Corresponding author

Correspondence to Zirui Dong.

Ethics declarations

Conflict of interest

The authors declare no conflicts of interest or competing interest.

Ethics approval

The study was approved by the institutional review boards of each collaborative site. Written consent for sample storage and genetic analyses of peripheral blood and invasive diagnostic samples was obtained from each participant.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 224 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Chau, M.H.K., Wang, H., Lai, Y. et al. Low-pass genome sequencing: a validated method in clinical cytogenetics. Hum Genet 139, 1403–1415 (2020). https://doi.org/10.1007/s00439-020-02185-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00439-020-02185-9

Search

Navigation