Skip to main content

Advertisement

SpringerLink
Log in

Next-generation molecular diagnosis: single-cell sequencing from bench to bedside

  • Review
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Single-cell sequencing (SCS) is a fast-growing, exciting field in genomic medicine. It enables the high-resolution study of cellular heterogeneity, and reveals the molecular basis of complicated systems, which facilitates the identification of new biomarkers for diagnosis and for targeting therapies. It also directly promotes the next generation of genomic medicine because of its ultra-high resolution and sensitivity that allows for the non-invasive and early detection of abnormalities, such as aneuploidy, chromosomal translocation, and single-gene disorders. This review provides an overview of the current progress and prospects for the diagnostic applications of SCS, specifically in pre-implantation genetic diagnosis/screening, non-invasive prenatal diagnosis, and analysis of circulating tumor cells. These analyses will accelerate the early and precise control of germline- or somatic-mutation-based diseases, particularly single-gene disorders, chromosome abnormalities, and cancers.

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

Similar content being viewed by others

References

  1. Novick A, Weiner M (1957) Enzyme induction as an all-or-none phenomenon. Proc Natl Acad Sci USA 43(7):553–566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Marinov GK, Williams BA, McCue K, Schroth GP, Gertz J, Myers RM, Wold BJ (2014) From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing. Genome Res 24(3):496–510. doi:10.1101/gr.161034.113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wilson NK, Kent DG, Buettner F, Shehata M, Macaulay IC, Calero-Nieto FJ, Sanchez Castillo M, Oedekoven CA, Diamanti E, Schulte R, Ponting CP, Voet T, Caldas C, Stingl J, Green AR, Theis FJ, Gottgens B (2015) Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations. Cell Stem Cell 16(6):712–724. doi:10.1016/j.stem.2015.04.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Coskun AF, Eser U, Islam S (2016) Cellular identity at the single-cell level. Mol BioSyst. doi:10.1039/c6mb00388e

    PubMed  Google Scholar 

  5. Speicher MR (2013) Single-cell analysis: toward the clinic. Genome Med 5(8):74. doi:10.1186/gm478

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Zhang X, Marjani SL, Hu Z, Weissman SM, Pan X, Wu S (2016) Single-cell sequencing for precise cancer research: progress and prospects. Cancer Res 76(6):1305–1312. doi:10.1158/0008-5472.can-15-1907

    Article  CAS  PubMed  Google Scholar 

  7. Van Loo P, Voet T (2014) Single cell analysis of cancer genomes. Curr Opin Genet Dev 24:82–91. doi:10.1016/j.gde.2013.12.004

    Article  PubMed  CAS  Google Scholar 

  8. Lu Y, Peng H, Jin Z, Cheng J, Wang S, Ma M, Lu Y, Han D, Yao Y, Li Y, Yuan H (2013) Preimplantation genetic diagnosis for a Chinese family with autosomal recessive Meckel–Gruber syndrome type 3 (MKS3). PLoS One 8(9):e73245. doi:10.1371/journal.pone.0073245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Di Naro E, Ghezzi F, Vitucci A, Tannoia N, Campanale D, D’Addario V, Holzgreve W, Hahn S (2000) Prenatal diagnosis of beta-thalassaemia using fetal erythroblasts enriched from maternal blood by a novel gradient. Mol Hum Reprod 6(6):571–574

    Article  PubMed  Google Scholar 

  10. Polzer B, Medoro G, Pasch S, Fontana F, Zorzino L, Pestka A, Andergassen U, Meier-Stiegen F, Czyz ZT, Alberter B, Treitschke S, Schamberger T, Sergio M, Bregola G, Doffini A, Gianni S, Calanca A, Signorini G, Bolognesi C, Hartmann A, Fasching PA, Sandri MT, Rack B, Fehm T, Giorgini G, Manaresi N, Klein CA (2014) Molecular profiling of single circulating tumor cells with diagnostic intention. EMBO Mol Med 6(11):1371–1386. doi:10.15252/emmm.201404033

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Miyamoto DT, Zheng Y, Wittner BS, Lee RJ, Zhu H, Broderick KT, Desai R, Fox DB, Brannigan BW, Trautwein J, Arora KS, Desai N, Dahl DM, Sequist LV, Smith MR, Kapur R, Wu CL, Shioda T, Ramaswamy S, Ting DT, Toner M, Maheswaran S, Haber DA (2015) RNA-Seq of single prostate CTCs implicates noncanonical Wnt signaling in antiandrogen resistance. Science (New York, NY) 349(6254):1351–1356. doi:10.1126/science.aab0917

    Article  CAS  Google Scholar 

  12. Inhorn MC, Patrizio P (2015) Infertility around the globe: new thinking on gender, reproductive technologies and global movements in the 21st century. Hum Reprod Update 21(4):411–426. doi:10.1093/humupd/dmv016

    Article  PubMed  Google Scholar 

  13. Chandra A, Copen CE, Stephen EH (2013) Infertility and impaired fecundity in the United States, 1982–2010: data from the National Survey of Family Growth. Natl Health Stat Rep (67):1–18 (11 p following 19)

  14. Hart RJ (2016) Physiological aspects of female fertility: role of the environment, modern lifestyle, and genetics. Physiol Rev 96(3):873–909. doi:10.1152/physrev.00023.2015

    Article  PubMed  Google Scholar 

  15. Wilkinson D, Schaefer GO, Tremellen K, Savulescu J (2015) Double trouble: should double embryo transfer be banned? Theor Med Bioeth 36(2):121–139. doi:10.1007/s11017-015-9324-x

    Article  PubMed  Google Scholar 

  16. Ning L, Li Z, Wang G, Hu W, Hou Q, Tong Y, Zhang M, Chen Y, Qin L, Chen X, Man HY, Liu P, He J (2015) Quantitative assessment of single-cell whole genome amplification methods for detecting copy number variation using hippocampal neurons. Sci Rep 5:11415. doi:10.1038/srep11415

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kumar A, Ryan A, Kitzman JO, Wemmer N, Snyder MW, Sigurjonsson S, Lee C, Banjevic M, Zarutskie PW, Lewis AP, Shendure J, Rabinowitz M (2015) Whole genome prediction for preimplantation genetic diagnosis. Genome Med 7(1):35. doi:10.1186/s13073-015-0160-4

    Article  PubMed  PubMed Central  Google Scholar 

  18. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, Bulletti C (2013) Genomic DNA in human blastocoele fluid. Reprod Biomed Online 26(6):603–610. doi:10.1016/j.rbmo.2013.02.012

    Article  CAS  PubMed  Google Scholar 

  19. Cohen J, Grudzinskas G, Johnson MH (2013) Embryonic DNA sampling without biopsy: the beginnings of non-invasive PGD? Reprod Biomed Online 26(6):520–521. doi:10.1016/j.rbmo.2013.03.001

    Article  CAS  PubMed  Google Scholar 

  20. Gianaroli L, Magli MC, Pomante A, Crivello AM, Cafueri G, Valerio M, Ferraretti AP (2014) Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril 102(6):1692–1699.e1696. doi:10.1016/j.fertnstert.2014.08.021

  21. Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, Peck AC, Sills ES, Salem RD (2012) Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 5(1):24. doi:10.1186/1755-8166-5-24

    Article  PubMed  PubMed Central  Google Scholar 

  22. Yang Z, Salem SA, Liu X, Kuang Y, Salem RD, Liu J (2013) Selection of euploid blastocysts for cryopreservation with array comparative genomic hybridization (aCGH) results in increased implantation rates in subsequent frozen and thawed embryo transfer cycles. Mol Cytogenet 6(1):32. doi:10.1186/1755-8166-6-32

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Ginsburg ES, Baker VL, Racowsky C, Wantman E, Goldfarb J, Stern JE (2011) Use of preimplantation genetic diagnosis and preimplantation genetic screening in the United States: a Society for Assisted Reproductive Technology Writing Group paper. Fertil Steril 96(4):865–868. doi:10.1016/j.fertnstert.2011.07.1139

    Article  PubMed  Google Scholar 

  24. Capalbo A, Ubaldi FM, Cimadomo D, Maggiulli R, Patassini C, Dusi L, Sanges F, Buffo L, Venturella R, Rienzi L (2016) Consistent and reproducible outcomes of blastocyst biopsy and aneuploidy screening across different biopsy practitioners: a multicentre study involving 2586 embryo biopsies. Hum Reprod (Oxford, England) 31(1):199–208. doi:10.1093/humrep/dev294

    Article  Google Scholar 

  25. Treff NR, Fedick A, Tao X, Devkota B, Taylor D, Scott RT, Jr. (2013) Evaluation of targeted next-generation sequencing-based preimplantation genetic diagnosis of monogenic disease. Fertil Steril 99(5):1377–1384.e1376. doi:10.1016/j.fertnstert.2012.12.018

  26. Yin X, Tan K, Vajta G, Jiang H, Tan Y, Zhang C, Chen F, Chen S, Zhang C, Pan X, Gong C, Li X, Lin C, Gao Y, Liang Y, Yi X, Mu F, Zhao L, Peng H, Xiong B, Zhang S, Cheng D, Lu G, Zhang X, Lin G, Wang W (2013) Massively parallel sequencing for chromosomal abnormality testing in trophectoderm cells of human blastocysts. Biol Reprod 88(3):69. doi:10.1095/biolreprod.112.106211

    Article  PubMed  CAS  Google Scholar 

  27. Findlay I, Ray P, Quirke P, Rutherford A, Lilford R (1995) Allelic drop-out and preferential amplification in single cells and human blastomeres: implications for preimplantation diagnosis of sex and cystic fibrosis. Hum Reprod (Oxford, England) 10(6):1609–1618

    Article  CAS  Google Scholar 

  28. Van der Aa N, Zamani Esteki M, Vermeesch JR, Voet T (2013) Preimplantation genetic diagnosis guided by single-cell genomics. Genome Med 5(8):71. doi:10.1186/gm475

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Gardner DK, Meseguer M, Rubio C, Treff NR (2015) Diagnosis of human preimplantation embryo viability. Hum Reprod Update 21(6):727–747. doi:10.1093/humupd/dmu064

    Article  PubMed  Google Scholar 

  30. Brezina PR, Kutteh WH (2015) Clinical applications of preimplantation genetic testing. BMJ (clinical research ed) 350:g7611. doi:10.1136/bmj.g7611

    Google Scholar 

  31. Wong KM, Repping S, Mastenbroek S (2014) Limitations of embryo selection methods. Semin Reprod Med 32(2):127–133. doi:10.1055/s-0033-1363554

    Article  PubMed  Google Scholar 

  32. Velilla E, Escudero T, Munne S (2002) Blastomere fixation techniques and risk of misdiagnosis for preimplantation genetic diagnosis of aneuploidy. Reprod Biomed Online 4(3):210–217

    Article  PubMed  Google Scholar 

  33. Treff NR, Tao X, Ferry KM, Su J, Taylor D, Scott RT Jr (2012) Development and validation of an accurate quantitative real-time polymerase chain reaction-based assay for human blastocyst comprehensive chromosomal aneuploidy screening. Fertil Steril 97(4):819–824. doi:10.1016/j.fertnstert.2012.01.115

    Article  CAS  PubMed  Google Scholar 

  34. Huang J, Zhao N, Wang X, Qiao J, Liu P (2015) Chromosomal characteristics at cleavage and blastocyst stages from the same embryos. J Assist Reprod Genet 32(5):781–787. doi:10.1007/s10815-015-0450-1

    Article  PubMed  PubMed Central  Google Scholar 

  35. Fiorentino F, Biricik A, Bono S, Spizzichino L, Cotroneo E, Cottone G, Kokocinski F, Michel CE (2014) Development and validation of a next-generation sequencing-based protocol for 24-chromosome aneuploidy screening of embryos. Fertil Steril 101(5):1375–1382. doi:10.1016/j.fertnstert.2014.01.051

    Article  CAS  PubMed  Google Scholar 

  36. Hellani A, Abu-Amero K, Azouri J, Al-Sharif H, Barblet H, El-Akoum S (2009) Pregnancy after preimplantation genetic diagnosis for brachydactyly type B. Reprod Biomed Online 18(1):127–131

    Article  PubMed  Google Scholar 

  37. Hellani A, Coskun S, Tbakhi A, Al-Hassan S (2005) Clinical application of multiple displacement amplification in preimplantation genetic diagnosis. Reprod Biomed Online 10(3):376–380

    Article  PubMed  Google Scholar 

  38. Wang L, Cram DS, Shen J, Wang X, Zhang J, Song Z, Xu G, Li N, Fan J, Wang S, Luo Y, Wang J, Yu L, Liu J, Yao Y (2014) Validation of copy number variation sequencing for detecting chromosome imbalances in human preimplantation embryos. Biol Reprod 91(2):37. doi:10.1095/biolreprod.114.120576

    Article  PubMed  CAS  Google Scholar 

  39. Wells D, Kaur K, Grifo J, Glassner M, Taylor JC, Fragouli E, Munne S (2014) Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J Med Genet 51(8):553–562. doi:10.1136/jmedgenet-2014-102497

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Li N, Wang L, Wang H, Ma M, Wang X, Li Y, Zhang W, Zhang J, Cram DS, Yao Y (2015) The performance of whole genome amplification methods and next-generation sequencing for pre-implantation genetic diagnosis of chromosomal abnormalities. J Genet Genom (Yi Chuan Xue Bao) 42(4):151–159. doi:10.1016/j.jgg.2015.03.001

    Article  Google Scholar 

  41. Hou Y, Fan W, Yan L, Li R, Lian Y, Huang J, Li J, Xu L, Tang F, Xie XS, Qiao J (2013) Genome analyses of single human oocytes. Cell 155(7):1492–1506. doi:10.1016/j.cell.2013.11.040

    Article  CAS  PubMed  Google Scholar 

  42. Lu S, Zong C, Fan W, Yang M, Li J, Chapman AR, Zhu P, Hu X, Xu L, Yan L, Bai F, Qiao J, Tang F, Li R, Xie XS (2012) Probing meiotic recombination and aneuploidy of single sperm cells by whole-genome sequencing. Science (New York, NY) 338(6114):1627–1630. doi:10.1126/science.1229112

    Article  CAS  Google Scholar 

  43. Huang J, Yan L, Fan W, Zhao N, Zhang Y, Tang F, Xie XS, Qiao J (2014) Validation of multiple annealing and looping-based amplification cycle sequencing for 24-chromosome aneuploidy screening of cleavage-stage embryos. Fertil Steril 102(6):1685–1691. doi:10.1016/j.fertnstert.2014.08.015

    Article  CAS  PubMed  Google Scholar 

  44. Yan L, Huang L, Xu L, Huang J, Ma F, Zhu X, Tang Y, Liu M, Lian Y, Liu P, Li R, Lu S, Tang F, Qiao J, Xie XS (2015) Live births after simultaneous avoidance of monogenic diseases and chromosome abnormality by next-generation sequencing with linkage analyses. Proc Natl Acad Sci USA 112(52):15964–15969. doi:10.1073/pnas.1523297113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Binder V, Bartenhagen C, Okpanyi V, Gombert M, Moehlendick B, Behrens B, Klein HU, Rieder H, Ida Krell PF, Dugas M, Stoecklein NH, Borkhardt A (2014) A new workflow for whole-genome sequencing of single human cells. Hum Mutat 35(10):1260–1270. doi:10.1002/humu.22625

    Article  CAS  PubMed  Google Scholar 

  46. Norwitz ER, Levy B (2013) Noninvasive prenatal testing: the future is now. Rev Obstet Gynecol 6(2):48–62

    PubMed  PubMed Central  Google Scholar 

  47. Lun FM, Chiu RW, Sun K, Leung TY, Jiang P, Chan KC, Sun H, Lo YM (2013) Noninvasive prenatal methylomic analysis by genomewide bisulfite sequencing of maternal plasma DNA. Clin Chem 59(11):1583–1594. doi:10.1373/clinchem.2013.212274

    Article  CAS  PubMed  Google Scholar 

  48. Chiu RW, Akolekar R, Zheng YW, Leung TY, Sun H, Chan KC, Lun FM, Go AT, Lau ET, To WW, Leung WC, Tang RY, Au-Yeung SK, Lam H, Kung YY, Zhang X, van Vugt JM, Minekawa R, Tang MH, Wang J, Oudejans CB, Lau TK, Nicolaides KH, Lo YM (2011) Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ (clinical research ed) 342:c7401. doi:10.1136/bmj.c7401

    Article  Google Scholar 

  49. Lau TK, Chen F, Pan X, Pooh RK, Jiang F, Li Y, Jiang H, Li X, Chen S, Zhang X (2012) Noninvasive prenatal diagnosis of common fetal chromosomal aneuploidies by maternal plasma DNA sequencing. J Matern Fetal Neonatal Med 25(8):1370–1374. doi:10.3109/14767058.2011.635730

    Article  CAS  PubMed  Google Scholar 

  50. Dan S, Wang W, Ren J, Li Y, Hu H, Xu Z, Lau TK, Xie J, Zhao W, Huang H, Xie J, Sun L, Zhang X, Wang W, Liao S, Qiang R, Cao J, Zhang Q, Zhou Y, Zhu H, Zhong M, Guo Y, Lin L, Gao Z, Yao H, Zhang H, Zhao L, Jiang F, Chen F, Jiang H, Li S, Li Y, Wang J, Wang J, Duan T, Su Y, Zhang X (2012) Clinical application of massively parallel sequencing-based prenatal noninvasive fetal trisomy test for trisomies 21 and 18 in 11,105 pregnancies with mixed risk factors. Prenat Diagn 32(13):1225–1232. doi:10.1002/pd.4002

    Article  PubMed  Google Scholar 

  51. Devaney SA, Palomaki GE, Scott JA, Bianchi DW (2011) Noninvasive fetal sex determination using cell-free fetal DNA: a systematic review and meta-analysis. JAMA 306(6):627–636. doi:10.1001/jama.2011.1114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Alfirevic Z, Sundberg K, Brigham S (2003) Amniocentesis and chorionic villus sampling for prenatal diagnosis. Cochrane Database Syst Rev (3):Cd003252. doi:10.1002/14651858.cd003252

  53. Ge Q, Li H, Yang Q, Lu J, Tu J, Bai Y, Lu Z (2011) Sequencing circulating miRNA in maternal plasma with modified library preparation. Clin Chim Acta Int J Clin Chem 412(21–22):1989–1994. doi:10.1016/j.cca.2011.07.010

  54. Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W, Evans MI, Dukes KA, Sullivan LM, Klinger KW, Bischoff FZ, Hahn S, Johnson KL, Lewis D, Wapner RJ, de la Cruz F (2002) Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diagn 22(7):609–615. doi:10.1002/pd.347

    Article  CAS  PubMed  Google Scholar 

  55. Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, Wainscoat JS, Johnson PJ, Chang AM, Hjelm NM (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 62(4):768–775. doi:10.1086/301800

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Daley R, Hill M, Chitty LS (2014) Non-invasive prenatal diagnosis: progress and potential. Arch Dis Child Fetal Neonatal Ed 99(5):F426–F430. doi:10.1136/archdischild-2013-304828

    Article  PubMed  Google Scholar 

  57. Choolani M, Mahyuddin AP, Hahn S (2012) The promise of fetal cells in maternal blood. Best Pract Res Clin Obstet Gynaecol 26(5):655–667. doi:10.1016/j.bpobgyn.2012.06.008

    Article  PubMed  Google Scholar 

  58. Grabar VV (2013) Prediction of complications of I trimester of pregnancy in women with distressed reproductive history by detection of transcervical trophoblastic cells. Georgian Med News 225:27–31

    Google Scholar 

  59. Mouawia H, Saker A, Jais JP, Benachi A, Bussieres L, Lacour B, Bonnefont JP, Frydman R, Simpson JL, Paterlini-Brechot P (2012) Circulating trophoblastic cells provide genetic diagnosis in 63 fetuses at risk for cystic fibrosis or spinal muscular atrophy. Reprod Biomed Online 25(5):508–520. doi:10.1016/j.rbmo.2012.08.002

    Article  PubMed  Google Scholar 

  60. Paterlini Brechot P, Mouawia H, Saker A (2011) Non-invasive prenatal diagnosis of cystic fibrosis. Arch Pediatr 18(1):111–118. doi:10.1016/j.arcped.2010.10.028

    Article  CAS  PubMed  Google Scholar 

  61. Zhang C, Zhang C, Chen S, Yin X, Pan X, Lin G, Tan Y, Tan K, Xu Z, Hu P, Li X, Chen F, Xu X, Li Y, Zhang X, Jiang H, Wang W (2013) A single cell level based method for copy number variation analysis by low coverage massively parallel sequencing. PLoS One 8(1):e54236. doi:10.1371/journal.pone.0054236

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Hua R, Barrett AN, Tan TZ, Huang Z, Mahyuddin AP, Ponnusamy S, Sandhu JS, Ho SS, Chan JK, Chong S, Quan S, Choolani M (2015) Detection of aneuploidy from single fetal nucleated red blood cells using whole genome sequencing. Prenat Diagn 35(7):637–644. doi:10.1002/pd.4491

    Article  CAS  PubMed  Google Scholar 

  63. Gregg AR, Gross SJ, Best RG, Monaghan KG, Bajaj K, Skotko BG, Thompson BH, Watson MS (2013) ACMG statement on noninvasive prenatal screening for fetal aneuploidy. Genet Med 15(5):395–398. doi:10.1038/gim.2013.29

    Article  CAS  PubMed  Google Scholar 

  64. Samango-Sprouse C, Banjevic M, Ryan A, Sigurjonsson S, Zimmermann B, Hill M, Hall MP, Westemeyer M, Saucier J, Demko Z, Rabinowitz M (2013) SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy. Prenat Diagn 33(7):643–649. doi:10.1002/pd.4159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Cardo L, Garcia BP, Alvarez FV (2010) Non-invasive fetal RHD genotyping in the first trimester of pregnancy. Clin Chem Lab Med 48(8):1121–1126. doi:10.1515/cclm.2010.234

    Article  CAS  PubMed  Google Scholar 

  66. Jensen TJ, Kim SK, Zhu Z, Chin C, Gebhard C, Lu T, Deciu C, van den Boom D, Ehrich M (2015) Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains. Genome Biol 16:78. doi:10.1186/s13059-015-0645-x

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  67. Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, Lau TK, Chim SS, Chung GT, Nicolaides KH, Lo YM (2006) Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 52(12):2211–2218. doi:10.1373/clinchem.2006.074997

    Article  CAS  PubMed  Google Scholar 

  68. Della Ragione F, Mastrovito P, Campanile C, Conti A, Papageorgiou EA, Hulten MA, Patsalis PC, Carter NP, D’Esposito M (2010) Differential DNA methylation as a tool for noninvasive prenatal diagnosis (NIPD) of X chromosome aneuploidies. J Mol Diagn 12(6):797–807. doi:10.2353/jmoldx.2010.090199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Vrba L, Jensen TJ, Garbe JC, Heimark RL, Cress AE, Dickinson S, Stampfer MR, Futscher BW (2010) Role for DNA methylation in the regulation of miR-200c and miR-141 expression in normal and cancer cells. PLoS One 5(1):e8697. doi:10.1371/journal.pone.0008697

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Cedar H, Bergman Y (2009) Linking DNA methylation and histone modification: patterns and paradigms. Nat Rev Genet 10(5):295–304. doi:10.1038/nrg2540

    Article  CAS  PubMed  Google Scholar 

  71. Wright CF, Burton H (2009) The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis. Hum Reprod Update 15(1):139–151. doi:10.1093/humupd/dmn047

    Article  CAS  PubMed  Google Scholar 

  72. Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM (1999) Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64(1):218–224. doi:10.1086/302205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Pfeifer I, Benachi A, Saker A, Bonnefont JP, Mouawia H, Broncy L, Frydman R, Brival ML, Lacour B, Dachez R, Paterlini-Brechot P (2016) Cervical trophoblasts for non-invasive single-cell genotyping and prenatal diagnosis. Placenta 37:56–60. doi:10.1016/j.placenta.2015.11.002

    Article  CAS  PubMed  Google Scholar 

  74. Boveri T (2008) Concerning the origin of malignant tumours by Theodor Boveri. Translated and annotated by Henry Harris. J Cell Sci 121 Suppl 1:1–84. doi:10.1242/jcs.025742

  75. Garraway LA, Lander ES (2013) Lessons from the cancer genome. Cell 153(1):17–37. doi:10.1016/j.cell.2013.03.002

    Article  CAS  PubMed  Google Scholar 

  76. Stratton MR (2011) Exploring the genomes of cancer cells: progress and promise. Science (New York, NY) 331(6024):1553–1558. doi:10.1126/science.1204040

    Article  CAS  Google Scholar 

  77. Stratton MR, Campbell PJ, Futreal PA (2009) The cancer genome. Nature 458(7239):719–724. doi:10.1038/nature07943

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Saunders NA, Simpson F, Thompson EW, Hill MM, Endo-Munoz L, Leggatt G, Minchin RF, Guminski A (2012) Role of intratumoural heterogeneity in cancer drug resistance: molecular and clinical perspectives. EMBO Mol Med 4(8):675–684. doi:10.1002/emmm.201101131

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Diaz LA Jr, Williams RT, Wu J, Kinde I, Hecht JR, Berlin J, Allen B, Bozic I, Reiter JG, Nowak MA, Kinzler KW, Oliner KS, Vogelstein B (2012) The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486(7404):537–540. doi:10.1038/nature11219

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Misale S, Yaeger R, Hobor S, Scala E, Janakiraman M, Liska D, Valtorta E, Schiavo R, Buscarino M, Siravegna G, Bencardino K, Cercek A, Chen CT, Veronese S, Zanon C, Sartore-Bianchi A, Gambacorta M, Gallicchio M, Vakiani E, Boscaro V, Medico E, Weiser M, Siena S, Di Nicolantonio F, Solit D, Bardelli A (2012) Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer. Nature 486(7404):532–536. doi:10.1038/nature11156

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Wheeler DA, Wang L (2013) From human genome to cancer genome: the first decade. Genome Res 23(7):1054–1062. doi:10.1101/gr.157602.113

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Navin NE (2015) Delineating cancer evolution with single-cell sequencing. Science Transl Med 7(296):296fs229. doi:10.1126/scitranslmed.aac8319

  83. Dalerba P, Kalisky T, Sahoo D, Rajendran PS, Rothenberg ME, Leyrat AA, Sim S, Okamoto J, Johnston DM, Qian D, Zabala M, Bueno J, Neff NF, Wang J, Shelton AA, Visser B, Hisamori S, Shimono Y, van de Wetering M, Clevers H, Clarke MF, Quake SR (2011) Single-cell dissection of transcriptional heterogeneity in human colon tumors. Nat Biotechnol 29(12):1120–1127. doi:10.1038/nbt.2038

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Jahan A, Yusuf MA, Loya A (2015) Fine-needle aspiration cytology in the diagnosis of pancreatic neuroendocrine tumors: a single-center experience of 25 cases. Acta Cytol 59(2):163–168. doi:10.1159/000381173

    Article  CAS  PubMed  Google Scholar 

  85. Roh MH (2015) The utilization of cytologic fine-needle aspirates of lung cancer for molecular diagnostic testing. J Pathol Transl Med 49(4):300–309. doi:10.4132/jptm.2015.06.16

    Article  PubMed  PubMed Central  Google Scholar 

  86. Li Y, Xu X, Song L, Hou Y, Li Z, Tsang S, Li F, Im KM, Wu K, Wu H, Ye X, Li G, Wang L, Zhang B, Liang J, Xie W, Wu R, Jiang H, Liu X, Yu C, Zheng H, Jian M, Nie L, Wan L, Shi M, Sun X, Tang A, Guo G, Gui Y, Cai Z, Li J, Wang W, Lu Z, Zhang X, Bolund L, Kristiansen K, Wang J, Yang H, Dean M, Wang J (2012) Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer. GigaScience 1(1):12. doi:10.1186/2047-217x-1-12

    Article  PubMed  PubMed Central  Google Scholar 

  87. Giorgadze TA, Scognamiglio T, Yang GC (2015) Fine-needle aspiration cytology of the solid variant of papillary thyroid carcinoma: a study of 13 cases with clinical, histologic, and ultrasound correlations. Cancer Cytopathol 123(2):71–81. doi:10.1002/cncy.21504

    Article  PubMed  Google Scholar 

  88. Lohr JG, Adalsteinsson VA, Cibulskis K, Choudhury AD, Rosenberg M, Cruz-Gordillo P, Francis JM, Zhang CZ, Shalek AK, Satija R, Trombetta JJ, Lu D, Tallapragada N, Tahirova N, Kim S, Blumenstiel B, Sougnez C, Lowe A, Wong B, Auclair D, Van Allen EM, Nakabayashi M, Lis RT, Lee GS, Li T, Chabot MS, Ly A, Taplin ME, Clancy TE, Loda M, Regev A, Meyerson M, Hahn WC, Kantoff PW, Golub TR, Getz G, Boehm JS (2014) Whole-exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer. Nat Biotechnol 32(5):479–484. doi:10.1038/nbt.2892

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Alix-Panabieres C, Pantel K (2014) Challenges in circulating tumour cell research. Nat Rev Cancer 14(9):623–631. doi:10.1038/nrc3820

    Article  CAS  PubMed  Google Scholar 

  90. Rhim AD, Thege FI, Santana SM, Lannin TB, Saha TN, Tsai S, Maggs LR, Kochman ML, Ginsberg GG, Lieb JG, Chandrasekhara V, Drebin JA, Ahmad N, Yang YX, Kirby BJ, Stanger BZ (2014) Detection of circulating pancreas epithelial cells in patients with pancreatic cystic lesions. Gastroenterology 146(3):647–651. doi:10.1053/j.gastro.2013.12.007

    Article  PubMed  Google Scholar 

  91. Chen CL, Mahalingam D, Osmulski P, Jadhav RR, Wang CM, Leach RJ, Chang TC, Weitman SD, Kumar AP, Sun L, Gaczynska ME, Thompson IM, Huang TH (2013) Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer. Prostate 73(8):813–826. doi:10.1002/pros.22625

    Article  PubMed  CAS  Google Scholar 

  92. Navin N, Hicks J (2011) Future medical applications of single-cell sequencing in cancer. Genome Med 3(5):31. doi:10.1186/gm247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. Brinkmann F, Hirtz M, Haller A, Gorges TM, Vellekoop MJ, Riethdorf S, Muller V, Pantel K, Fuchs H (2015) A versatile microarray platform for capturing rare cells. Sci Rep 5:15342. doi:10.1038/srep15342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Zhou J, Ren F, Wu W, Zhang S, Xiao X, Xu J, Jiang C (2012) Controllable synthesis and catalysis application of hierarchical PS/Au core-shell nanocomposites. J Colloid Interface Sci 387(1):47–55. doi:10.1016/j.jcis.2012.07.093

    Article  CAS  PubMed  Google Scholar 

  95. Ren SC, Qu M, Sun YH (2013) Investigating intratumour heterogeneity by single-cell sequencing. Asian J Androl 15(6):729–734. doi:10.1038/aja.2013.106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Adalsteinsson VA, Love JC (2014) Towards engineered processes for sequencing-based analysis of single circulating tumor cells. Curr Opinion Chem Eng 4:97–104. doi:10.1016/j.coche.2014.01.011

    Article  Google Scholar 

  97. Moller EK, Kumar P, Voet T, Peterson A, Van Loo P, Mathiesen RR, Fjelldal R, Grundstad J, Borgen E, Baumbusch LO, Naume B, Borresen-Dale AL, White KP, Nord S, Kristensen VN (2013) Next-generation sequencing of disseminated tumor cells. Front Oncol 3:320. doi:10.3389/fonc.2013.00320

    Article  PubMed  PubMed Central  Google Scholar 

  98. Navin NE (2014) Cancer genomics: one cell at a time. Genome Biol 15(8):452. doi:10.1186/s13059-014-0452-9

    Article  PubMed  PubMed Central  Google Scholar 

  99. Yu C, Yu J, Yao X, Wu WK, Lu Y, Tang S, Li X, Bao L, Li X, Hou Y, Wu R, Jian M, Chen R, Zhang F, Xu L, Fan F, He J, Liang Q, Wang H, Hu X, He M, Zhang X, Zheng H, Li Q, Wu H, Chen Y, Yang X, Zhu S, Xu X, Yang H, Wang J, Zhang X, Sung JJ, Li Y, Wang J (2014) Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing. Cell Res 24(6):701–712. doi:10.1038/cr.2014.43

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Hou Y, Song L, Zhu P, Zhang B, Tao Y, Xu X, Li F, Wu K, Liang J, Shao D, Wu H, Ye X, Ye C, Wu R, Jian M, Chen Y, Xie W, Zhang R, Chen L, Liu X, Yao X, Zheng H, Yu C, Li Q, Gong Z, Mao M, Yang X, Yang L, Li J, Wang W, Lu Z, Gu N, Laurie G, Bolund L, Kristiansen K, Wang J, Yang H, Li Y, Zhang X, Wang J (2012) Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell 148(5):873–885. doi:10.1016/j.cell.2012.02.028

    Article  CAS  PubMed  Google Scholar 

  101. Ni X, Zhuo M, Su Z, Duan J, Gao Y, Wang Z, Zong C, Bai H, Chapman AR, Zhao J, Xu L, An T, Ma Q, Wang Y, Wu M, Sun Y, Wang S, Li Z, Yang X, Yong J, Su XD, Lu Y, Bai F, Xie XS, Wang J (2013) Reproducible copy number variation patterns among single circulating tumor cells of lung cancer patients. Proc Natl Acad Sci USA 110(52):21083–21088. doi:10.1073/pnas.1320659110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Dago AE, Stepansky A, Carlsson A, Luttgen M, Kendall J, Baslan T, Kolatkar A, Wigler M, Bethel K, Gross ME, Hicks J, Kuhn P (2014) Rapid phenotypic and genomic change in response to therapeutic pressure in prostate cancer inferred by high content analysis of single circulating tumor cells. PLoS One 9(8):e101777. doi:10.1371/journal.pone.0101777

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  103. Navin N, Kendall J, Troge J, Andrews P, Rodgers L, McIndoo J, Cook K, Stepansky A, Levy D, Esposito D, Muthuswamy L, Krasnitz A, McCombie WR, Hicks J, Wigler M (2011) Tumour evolution inferred by single-cell sequencing. Nature 472(7341):90–94. doi:10.1038/nature09807

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Suzuki A, Matsushima K, Makinoshima H, Sugano S, Kohno T, Tsuchihara K, Suzuki Y (2015) Single-cell analysis of lung adenocarcinoma cell lines reveals diverse expression patterns of individual cells invoked by a molecular target drug treatment. Genome Biol 16:66. doi:10.1186/s13059-015-0636-y

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  105. Roukos DH (2010) Novel clinico-genome network modeling for revolutionizing genotype-phenotype-based personalized cancer care. Expert Rev Mol Diagn 10(1):33–48. doi:10.1586/erm.09.69

    Article  CAS  PubMed  Google Scholar 

  106. Yan L, Yang M, Guo H, Yang L, Wu J, Li R, Liu P, Lian Y, Zheng X, Yan J, Huang J, Li M, Wu X, Wen L, Lao K, Li R, Qiao J, Tang F (2013) Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells. Nat Struct Mol Biol 20(9):1131–1139. doi:10.1038/nsmb.2660

    Article  CAS  PubMed  Google Scholar 

  107. Zhu Z, Wang DC, Popescu LM, Wang X (2014) Single-cell transcriptome in the identification of disease biomarkers: opportunities and challenges. J Transl Med 12:212. doi:10.1186/s12967-014-0212-3

    Article  PubMed  PubMed Central  Google Scholar 

  108. Kantlehner M, Kirchner R, Hartmann P, Ellwart JW, Alunni-Fabbroni M, Schumacher A (2011) A high-throughput DNA methylation analysis of a single cell. Nucleic Acids Res 39(7):e44. doi:10.1093/nar/gkq1357

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Soden SE, Farrow EG, Saunders CJ, Lantos JD (2012) Genomic medicine: evolving science, evolving ethics. Pers Med 9(5):523–528. doi:10.2217/pme.12.56

    Article  CAS  Google Scholar 

  110. Eun JR, Jung YJ, Zhang Y, Zhang Y, Tschudy-Seney B, Ramsamooj R, Wan YJ, Theise ND, Zern MA, Duan Y (2014) Hepatoma SK Hep-1 cells exhibit characteristics of oncogenic mesenchymal stem cells with highly metastatic capacity. PLoS One 9(10):e110744. doi:10.1371/journal.pone.0110744

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  111. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA (2009) mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 6(5):377–382. doi:10.1038/nmeth.1315

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Drs. Sherman Weissman and Charles Wang for their valuable comments and suggestions. This work was supported by the Southern Medical University (C1033267), the National Natural Science Foundation of China (No. 81402529), Zhejiang Provincial Foundation for Natural Sciences (No. LZ15H220001), Zhejiang Science and Technology Planning Project of Health & Medicine (No. 2015PYA009) and Hangzhou Science and Technology Development Program (No. 20150733Q63), and the US National Institutes of Health Grants, 1P01GM099130-01 and R01DK100858. The authors declare no conflict of interest.

Author information

Author notes

    Authors and Affiliations

    1. Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Guangdong Province Key Laboratory of Biochip Technology, Southern Medical University, Guangzhou, 510515, Guangdong, People’s Republic of China

      Xinghua Pan

    2. Department of Genetics, School of Medicine, Yale University, New Haven, CT, 06520, USA

      Wanjun Zhu, Jialing Zhang & Xinghua Pan

    3. Hangzhou Cancer Institution, Hangzhou Cancer Hospital, Hangzhou, 310002, Zhejiang, People’s Republic of China

      Xiao-Yan Zhang & Shixiu Wu

    4. Department of Biology, Central Connecticut State University, New Britain, CT, 06050, USA

      Sadie L. Marjani

    5. Precision Medicine Center, West China Hospital, Sichuan University, Chengdu, 610041, Sichuan, China

      Wengeng Zhang

    6. College of Veterinary Medicine, University of Minnesota, Twin Cities, Saint Paul, MN, 55108, USA

      Wanjun Zhu

    Authors
    1. Wanjun Zhu
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Xiao-Yan Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Sadie L. Marjani
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Jialing Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Wengeng Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Shixiu Wu
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Xinghua Pan
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding authors

    Correspondence to Shixiu Wu or Xinghua Pan.

    Additional information

    W. Zhu and X.-Y. Zhang contributed equally to this work.

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Zhu, W., Zhang, XY., Marjani, S.L. et al. Next-generation molecular diagnosis: single-cell sequencing from bench to bedside. Cell. Mol. Life Sci. 74, 869–880 (2017). https://doi.org/10.1007/s00018-016-2368-x

    Download citation

    • Received:

    • Revised:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s00018-016-2368-x

    Keywords

    Search

    Navigation