单细胞测序在表观遗传学中的应用

1、单细胞测序在表观遗传学中的应用单细胞测序（single cell sequencing）是指先分离纯化单个细胞，并提取该细胞的DNA或RNA进行扩增后测序的技术 HYPERLINK l _ENREF_1 o Levsky, 2002 #1 ADDIN EN.CITE ADDIN EN.CITE.DATA 1。该技术起步虽不太久，但已深受关注，被竞相用于研发和生产、医用 HYPERLINK l _ENREF_2 o Wen, 2016 #2 ADDIN EN.CITE Wen2016222217Wen, L.Tang, F.Biodynamic Optical Imaging Center, C

2、ollege of Life Sciences, Peking University, Beijing, 100871, China. wenlu.Biodynamic Optical Imaging Center, College of Life Sciences, Peking University, Beijing, 100871, China. tangfuchou.Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China. tangfuchou.Single-cell seq

3、uencing in stem cell biologyGenome BiolGenome biologyGenome BiolGenome biologyGenome BiolGenome biology71172016Apr 151474-760X (Electronic)1474-7596 (Linking)27083874/pubmed/27083874483254010.1186/s13059-016-0941-02。相比于宏观组织样品的测序，单细胞测序对于细胞和组织的异质性具有更加明确的分辩，当然，难度也更高。由于在某些组织或研究领域（如癌症、干细胞池等），细胞处于显著的动态发展中

4、，细胞与细胞间差异巨大，而后续的显著变化（如成瘤、分化等）、组织的异质性，往往来源一个细胞克隆；而另一方面，利用表观遗传学（epigenetics）这种非基因组核酸序列的功能差异，研究某一组织生理病理变化，具有极大的必要性 HYPERLINK l _ENREF_3 o Stary, 2015 #4 ADDIN EN.CITE Stary2015434417Stary, C. M.Patel, H. H.Roth, D. M.From the Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford Univ

5、ersity School of Medicine, Stanford, California (C.M.S.); and Department of Anesthesiology, Veterans Affairs San Diego Healthcare System, University of California, San Diego, La Jolla, California (H.H.P., D.M.R.).Epigenetics: The Epicenter for Future Anesthesia Research?AnesthesiologyAnesthesiologyA

6、nesthesiologyAnesthesiologyAnesthesiologyAnesthesiology743-41234AnimalsIsoflurane/*administration & dosageMaleMicroRNAs/*physiologyMitochondrial Membrane Transport Proteins/*metabolismMyocardial Reperfusion Injury/*metabolismNitric Oxide Synthase/*metabolismProto-Oncogene Proteins c-akt/*metabolism2

7、015Oct1528-1175 (Electronic)0003-3022 (Linking)26259141/pubmed/2625914110.1097/ALN.00000000000008083。此时，如果笼统考察组织块的表观遗传学改变，很容易模糊了幂律分布下细胞水平的差异 HYPERLINK l _ENREF_4 o Hyun, 2015 #5 ADDIN EN.CITE Hyun2015545517Hyun, B. R.McElwee, J. L.Soloway, P. D.Division of Nutritional Sciences, Cornell University, I

8、thaca, NY 14853, USA.Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA. Electronic address: soloway.Single molecule and single cell epigenomicsMethodsMethodsMethodsMethodsMethodsMethods41-5072Chromatin Immunoprecipitation/methodsDNA MethylationEpigenomics/*methodsMass Spect

9、rometry/methodsMicrofluidics/methodsSingle-Cell Analysis/*methodsSoftwareWorkflow2015Jan 151095-9130 (Electronic)1046-2023 (Linking)25204781/pubmed/25204781430026610.1016/j.ymeth.2014.08.0154。相反，结合单细胞测序技术，可着眼于癌症、干细胞等早期的表观遗传学变化，对于理解病因、早期诊断、晚期控制极具价值。尽管分离纯化单细胞难度较高，而且DNA的表观遗传修饰很难通过传统的聚合酶扩增（通常表观遗传学测序方法包括

10、BS-seq和ChIP-seq），因此，单细胞测序用于表观遗传学分析，在技术上极具挑战 HYPERLINK l _ENREF_5 o Wang, 2015 #6 ADDIN EN.CITE Wang2015656617Wang, Y.Navin, N. E.Department of Genetics, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA.Department of Genetics, University of Texas, MD Anderson Cancer Center, Hous

11、ton, TX 77030, USA; Department of Bioinformatics and Computational Biology, University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address: nnavin.Advances and applications of single-cell sequencing technologiesMol CellMolecular cellMol CellMolecular cellMol CellMolecular

12、 cell598-609584AnimalsBiomedical Research/*methods/trendsForecastingGenomics/*methodsHigh-Throughput Nucleotide Sequencing/*methodsHumansSingle-Cell Analysis/*methods2015May 211097-4164 (Electronic)1097-2765 (Linking)26000845/pubmed/26000845444195410.1016/j.molcel.2015.05.0055。但目前已有研究者在不同领域做出了可喜进展。例

13、如，基于BS-seq的方法中，Guo等利用一种reduced representation bisulfite sequencing (scRRBS)方法，可在一个小鼠胚胎干细胞中的整个基因组范围内，灵敏地检测高达150万个CpG位点，并用该方法进行了单细胞表观遗传测序。该方法用于肿瘤发生、胚胎发育等领域，在各时间点DNA甲基化的动态变化，极具前景 HYPERLINK l _ENREF_6 o Guo, 2013 #7 ADDIN EN.CITE ADDIN EN.CITE.DATA 6。再如，基于ChIP-seq的方法中，Assaf Rotem等结合微流控和DNA特征码技术进行了数千个单细胞

14、测序，观察了单细胞水平上传统转录分析无法获取的表观遗传学异质性 HYPERLINK l _ENREF_7 o Rotem, 2015 #8 ADDIN EN.CITE ADDIN EN.CITE.DATA 7。此外，Kantlehner等基于甲基化敏感的内切酶的方法，对肿瘤细胞进行了高通量单细胞测序，分析了单细胞间感兴趣基因的甲基化差异 HYPERLINK l _ENREF_8 o Kantlehner, 2011 #11 ADDIN EN.CITE Kantlehner2011118111117Kantlehner, M.Kirchner, R.Hartmann, P.Ellwart, J.

15、 W.Alunni-Fabbroni, M.Schumacher, A.Beckman Coulter Biomedical GmbH, Advalytix Products, Munich, Germany.A high-throughput DNA methylation analysis of a single cellNucleic Acids ResNucleic acids researchNucleic Acids ResNucleic acids researchNucleic Acids ResNucleic acids researche44397Cell LineCell

16、 Line, TumorCpG Islands*DNA MethylationDNA Restriction EnzymesHigh-Throughput Screening AssaysHumansLymphocytes/metabolismPolymerase Chain Reaction*Single-Cell Analysis2011Apr1362-4962 (Electronic)0305-1048 (Linking)21266484/pubmed/21266484307415810.1093/nar/gkq13578。最近一项研究，由Christof Angermueller等发表

17、，更将此技术的应用做出了巨大推进。他们在61个胚胎干细胞上，利用一种平行的单细胞全基因组甲基化测序和转录组测序的联合技术，从全基因组水平分析了DNA甲基化异质性和转录异质性的关联 HYPERLINK l _ENREF_9 o Angermueller, 2016 #9 ADDIN EN.CITE ADDIN EN.CITE.DATA 9。Darren A. Cusanovich等利用大量的单细胞，分别进行单细胞测序观测了染色质的可接近性，来阐释组织的异质性 HYPERLINK l _ENREF_10 o Cusanovich, 2015 #3 ADDIN EN.CITE ADDIN EN.CI

18、TE.DATA 10。Lorthongpanich等利用单细胞测序甲基化分析，观察了人类早期胚胎的表观遗传学嵌合现象，并用以预测胚胎后期的生理缺陷，该研究更进一步为将单细胞表观遗传学测序用于孕产的临床诊断和治疗做了铺垫 HYPERLINK l _ENREF_11 o Lorthongpanich, 2013 #12 ADDIN EN.CITE ADDIN EN.CITE.DATA 11。以上研究表明，单细胞测序结合表观遗传视角的应用，已逐步向着更高的分辩能力、灵敏度、和更多的维度迈进。相信在以肿瘤研究、干细胞研究为代表的前沿热门领域，单细胞测序用于表观遗传学极具前景、并有丰富的进一步发展空间

19、HYPERLINK l _ENREF_12 o Ye, 2016 #13 ADDIN EN.CITE ADDIN EN.CITE.DATA 12。参考文献 ADDIN EN.REFLIST 1.Levsky JM, Shenoy SM, Pezo RC, Singer RH. Single-cell gene expression profiling. Science 2002; 297(5582): 836-40.2.Wen L, Tang F. Single-cell sequencing in stem cell biology. Genome biology 2016; 17: 71.

20、3.Stary CM, Patel HH, Roth DM. Epigenetics: The Epicenter for Future Anesthesia Research? Anesthesiology 2015; 123(4): 743-4.4.Hyun BR, McElwee JL, Soloway PD. Single molecule and single cell epigenomics. Methods 2015; 72: 41-50.5.Wang Y, Navin NE. Advances and applications of single-cell sequencing

21、 technologies. Molecular cell 2015; 58(4): 598-609.6.Guo H, Zhu P, Wu X, Li X, Wen L, Tang F. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome research 2013; 23(12): 2126-35.7.Rotem A, Ram O, Shoresh N, et al. Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state. Nature biotechnology 2015; 33(11): 1165-72.8.Kantlehner M, Kirchner R, Hartmann P, Ellwart JW, Alunni-Fabbroni M, Schumacher A. A high-throughput DNA methylation analysis of a sin