单细胞转录组测序技术原理及其应用
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摘要
基因转录调控及其机制分析是后基因组时代生物学研究的重点之一。常规研究往往建立在细胞群体或者组织上,一般表现出的是细胞的综合反应,难以判断单个细胞的情况,而在肿瘤、疾病预防等方面都需要对少量或单个细胞进行研究,因此,单细胞研究技术必不可少。常规的基因组扩增技术难以消除内含子的影响,并且无法用来阐述某些类型或某些特殊单个细胞之间基因表达的异质性,而转录组测序从RNA入手,将其反转录为cDNA后再进行大量扩增,可以完美地消除内含子的影响。本文主要就单细胞分离技术、单细胞转录组测序技术、高通量测序技术进行综述,并列举了现阶段的研究进展,分析了该项技术在各个领域的应用以及在家畜育种中的应用前景。
Gene transcriptional regulation and its mechanism analysis are one of the key points in post-genome biology research.Conventional research is often based on cell populations or tissues,showing cell synthesis reaction.It is difficult to judge the condition of single cells.In terms of tumors,disease prevention,etc.,it is necessary to conduct research on a small number of cells or a single cell.Therefore,a single cell research technique is essential.Conventional genome amplification technology is difficult to eliminate the influence of introns,and cannot be used to expound the heterogeneity of gene expression between certain types or special single cell.Transcriptome sequencing begins with RNA,and reverse transcription of RNA into cDNA followed by extensive amplification can eliminate the effects of introns perfectly.This article mainly summarized single-celled separation technology,single-cell transcriptome sequencing technology,and high-throughput sequencing technology.At the same time,we listed some of the research progresses in this area,and analyzed the application of the technology in various fields and the application prospect of animal breeding.
Gene transcriptional regulation and its mechanism analysis are one of the key points in post-genome biology research.Conventional research is often based on cell populations or tissues,showing cell synthesis reaction.It is difficult to judge the condition of single cells.In terms of tumors,disease prevention,etc.,it is necessary to conduct research on a small number of cells or a single cell.Therefore,a single cell research technique is essential.Conventional genome amplification technology is difficult to eliminate the influence of introns,and cannot be used to expound the heterogeneity of gene expression between certain types or special single cell.Transcriptome sequencing begins with RNA,and reverse transcription of RNA into cDNA followed by extensive amplification can eliminate the effects of introns perfectly.This article mainly summarized single-celled separation technology,single-cell transcriptome sequencing technology,and high-throughput sequencing technology.At the same time,we listed some of the research progresses in this area,and analyzed the application of the technology in various fields and the application prospect of animal breeding.
引文
[1]朱翔,浦春,武其文.单细胞转录组测序的方法原理及应用[J].分子诊断与治疗杂志,2016,21(1):1-65.
[2]杜璐,王少军,彭广华.单细胞转录组测序数据分析算法在干细胞研究中的应用[J].现代生物医学进展,2018,18(1):155-159.
[3]Tang F,Barbacior u C,Wang Y,et al.mRNA-Seq wholetranscriptome analysis of a single cell[J].Nat Methods,20096(5):377-382.
[4]Moignard V,Macaulay I C,Swiers G,et al.Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis[J].Nat Cell Biol,2013,15(4):363-372.
[5]Tang F,Barbacioru C,Bao S,et al.Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis[J].Cell Stem Cell,2010,6(5):468-478.
[6]庄琪琛,宁芮之,麻远,等.微流控技术应用于细胞分析的研究进展[J].分析化学,2016,3(4):522-532.
[7]Streets A M,Zhang X,Cao C,et al.Microf luidic single-cell whole-transcriptome sequencing[J].Proc Natl Acad Sci USA2014,111(19):7048-7053
[8]Wu A R,Kawahara T L,Rapicavoli N A,et al.High throug hput automated ch romat i n i m mu noprecipit at ion a s a platform for drug screening and antibody validation[J].Lab Chip,2012,12(12):2190-2198.
[9]Reizel Y,Chapal-Ilani N,Adar R,et al.Colon stem cell and crypt dynamics exposed by cell lineage reconstruction[J].PLoS Genet,2011,7(7):e1002192
[10]Rota L M,Lazzarino D A,Ziegler A N,et al.Determining mam-mosphere-forming potential:application of the limiting dilution analysis[J].J Mammary Gland Biol,2012,17(2):119-123.
[11]纪艳丽,彭广华.单细胞转录组测序在眼科研究领域中的应用[J].眼科新进展,2018,38(1):84-88.
[12]Kirkness E F,Grindberg R V,Yee-Greenbaum J,et al.Sequencing of isolated sperm cells for direct haplotyping of a human genome[J].Genome Res,2013,23(5):826-832.
[13]Shapiro E,Biezuner T,Linnarsson S,et al.Single-cell sequencing-based technologies will revolutionize whole-organism science[J].Nat Rev Genet,2013,14(9):618-630.
[14]Zhang X,Marjani S L,Hu Z,et al.Single-Cell Sequencing for precise cancer research:progress and prospects[J].Cancer Res,2016,76(6):1305-1312.
[15]贾昌路,张瑶,朱玲,等.转录组测序技术在生物测序中的应用研究进展[J].分子植物育种,2015,13(10):2388-2394.
[16]Wang Y,Navin N E.Advances and applications of single-cell sequencing technologies[J].Mol Cell,2015,58(4):598-609.
[17]Kurimoto K,Yabuta Y,Ohinata Y,et al.An improved singlecell cDNA amplification method for efficient high-density oligonucleotidemicroarray analysis[J].Nucleic Acids Res,2006,34(5):e42.
[18]Iscove N N,Barbara M,Gu M,et al.Represent ation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA[J].Nat Biotechnol2002,20(9):940-943.
[19]Ramsk?ld D,Luo S,Wang Y C,et al.Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells[J].Nat Biotechnol,2012,30(8):777-782.
[20]Goetz J J,Trimarchi J M.Transcriptome sequencing of single cells with Smart-Seq[J].Nat Biotechnol,2012,30(8):763-765.
[21]Picelli S,Bj?rklund?K,Faridani O R,et al.Smart-seq2 for sensitive full-length transcriptome profiling in single cells[J]Nat Methods,2013,10(11):1096-1098.
[22]Kanter I,Kalisky T.Single cell transcriptomics:methods and applications[J].Front Oncol,2015,5:53.
[23]薛瑞栋,李若岩,白凡.单细胞测序:技术,应用和未来发展(英文)[J].Sci Bull,2015,1:33-42,2.
[24]Islam S,Zeisel A,Joost S,et al.Quantitative single-cell RNA-seq with unique molecular identifiers[J].Nat Methods,2014,11(2):163-166.
[25]Macosko E Z,Basu A,Satija R,et al.Highly parallel genomewide expression profiling of individual cells using nanoliter droplets[J].Cell,2015,161(5):1202-1214.
[26]Zilionis R,Nainys J,Veres A,et al.Single-cell barcoding and sequencing using droplet microf luidics[J].Nat Protoc,201712(1):44-73.
[27]刘维瑜,金春莲.多重置换扩增--一种新的全基因组扩增技术[J].国际遗传学杂志,2007,30(4):265-268.
[28]唐琴,唐秀华,孙威江.转录组学技术及其在茶树研究中的应用[J].天然产物研究与开发,2018(5):1-12.
[29]祁云霞,刘永斌,荣威恒.转录组研究新技术:RNA-Seq及其应用[J].遗传,2011,33(11):1191-1202.
[30]Wang J,Song Y.Single cell sequencing:a distinct new field[J].Clin Transl Med,2017,6(1):10.
[31]K i m K T,Lee H W,Lee H O,et al.Si ngle-cell m R NAsequencing identifies subclonal heterogeneity in anti-cancer drug responses of lung adenocarcinoma cells[J].Genome Biol,2015,16:127.
[32]Eirew P,Steif A,Khattra J,et al.Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution[J]Nature,2015,518(7539):422-426.
[33]Yu C,Yu J,Yao X,et al.Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing[J]Cell Res,2014,24(6):701-712.
[34]Li Y,Xu X,Song L,et al.Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer[J].Gigascience,2012,1(1):12.
[35]Hughes A E,Magrini V,Demeter R,et al.Clonal architecture of secondary acute myeloid leukemia defined by single-cell sequencing[J].PLoS Genet,2014,10(7):e1004462.
[36]Ramsk?ld D,Luo S,Wang Y C,et al.Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells[J].Nat Biotechnol,2012,30(8):777-782.
[37]Goetz J J,Trimarchi J M.Single-cell profiling of developing and mature retinal neurons[J].J Vis Exp,2012,62(62):e3971-e3824.
[38]Shekhar K,Lapan S W,Whitney I E,et al.Comprehensive classification of retinal bipolar neurons by single-cell transcriptomics[J].Cell,2016,166(5):1308-1323.
[39]Mahata B,Zhang X,Kolodziejczyk A A,et al.Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis[J].Cell Rep,2014,7(4):1130-1142.
[40]Shalek A K,Satija R,Adiconis X,et al.Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells[J].Nature,2013,498(7453):236-240.
[41]蔡缪荧.羔羊JIVET相关技术及影响其卵母细胞发育功能基因的研究[D].扬州:扬州大学,2015.
[42]Di R,He J,Song S,et al.Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season[J].BMC Genomics,2014,15:899.
[43]王娟红,臧明,郑毛亮,等.绵羊繁殖调控的转录组学和蛋白组学研究进展[J].中国草食动物科学,2018,38(1):49-52.
[44]兰道亮,熊显荣,陈亚冰,等.牦牛体外成熟卵母细胞差异转录组学研究[J].中国科学:生命科学,2017,47(10):1099-1112.
[45]兰道亮,熊显荣,柴志欣,等.牦牛发情期卵巢比较转录组学研究[J].畜牧兽医学报,2016,47(9):1830-1839.
[2]杜璐,王少军,彭广华.单细胞转录组测序数据分析算法在干细胞研究中的应用[J].现代生物医学进展,2018,18(1):155-159.
[3]Tang F,Barbacior u C,Wang Y,et al.mRNA-Seq wholetranscriptome analysis of a single cell[J].Nat Methods,20096(5):377-382.
[4]Moignard V,Macaulay I C,Swiers G,et al.Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis[J].Nat Cell Biol,2013,15(4):363-372.
[5]Tang F,Barbacioru C,Bao S,et al.Tracing the derivation of embryonic stem cells from the inner cell mass by single-cell RNA-Seq analysis[J].Cell Stem Cell,2010,6(5):468-478.
[6]庄琪琛,宁芮之,麻远,等.微流控技术应用于细胞分析的研究进展[J].分析化学,2016,3(4):522-532.
[7]Streets A M,Zhang X,Cao C,et al.Microf luidic single-cell whole-transcriptome sequencing[J].Proc Natl Acad Sci USA2014,111(19):7048-7053
[8]Wu A R,Kawahara T L,Rapicavoli N A,et al.High throug hput automated ch romat i n i m mu noprecipit at ion a s a platform for drug screening and antibody validation[J].Lab Chip,2012,12(12):2190-2198.
[9]Reizel Y,Chapal-Ilani N,Adar R,et al.Colon stem cell and crypt dynamics exposed by cell lineage reconstruction[J].PLoS Genet,2011,7(7):e1002192
[10]Rota L M,Lazzarino D A,Ziegler A N,et al.Determining mam-mosphere-forming potential:application of the limiting dilution analysis[J].J Mammary Gland Biol,2012,17(2):119-123.
[11]纪艳丽,彭广华.单细胞转录组测序在眼科研究领域中的应用[J].眼科新进展,2018,38(1):84-88.
[12]Kirkness E F,Grindberg R V,Yee-Greenbaum J,et al.Sequencing of isolated sperm cells for direct haplotyping of a human genome[J].Genome Res,2013,23(5):826-832.
[13]Shapiro E,Biezuner T,Linnarsson S,et al.Single-cell sequencing-based technologies will revolutionize whole-organism science[J].Nat Rev Genet,2013,14(9):618-630.
[14]Zhang X,Marjani S L,Hu Z,et al.Single-Cell Sequencing for precise cancer research:progress and prospects[J].Cancer Res,2016,76(6):1305-1312.
[15]贾昌路,张瑶,朱玲,等.转录组测序技术在生物测序中的应用研究进展[J].分子植物育种,2015,13(10):2388-2394.
[16]Wang Y,Navin N E.Advances and applications of single-cell sequencing technologies[J].Mol Cell,2015,58(4):598-609.
[17]Kurimoto K,Yabuta Y,Ohinata Y,et al.An improved singlecell cDNA amplification method for efficient high-density oligonucleotidemicroarray analysis[J].Nucleic Acids Res,2006,34(5):e42.
[18]Iscove N N,Barbara M,Gu M,et al.Represent ation is faithfully preserved in global cDNA amplified exponentially from sub-picogram quantities of mRNA[J].Nat Biotechnol2002,20(9):940-943.
[19]Ramsk?ld D,Luo S,Wang Y C,et al.Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells[J].Nat Biotechnol,2012,30(8):777-782.
[20]Goetz J J,Trimarchi J M.Transcriptome sequencing of single cells with Smart-Seq[J].Nat Biotechnol,2012,30(8):763-765.
[21]Picelli S,Bj?rklund?K,Faridani O R,et al.Smart-seq2 for sensitive full-length transcriptome profiling in single cells[J]Nat Methods,2013,10(11):1096-1098.
[22]Kanter I,Kalisky T.Single cell transcriptomics:methods and applications[J].Front Oncol,2015,5:53.
[23]薛瑞栋,李若岩,白凡.单细胞测序:技术,应用和未来发展(英文)[J].Sci Bull,2015,1:33-42,2.
[24]Islam S,Zeisel A,Joost S,et al.Quantitative single-cell RNA-seq with unique molecular identifiers[J].Nat Methods,2014,11(2):163-166.
[25]Macosko E Z,Basu A,Satija R,et al.Highly parallel genomewide expression profiling of individual cells using nanoliter droplets[J].Cell,2015,161(5):1202-1214.
[26]Zilionis R,Nainys J,Veres A,et al.Single-cell barcoding and sequencing using droplet microf luidics[J].Nat Protoc,201712(1):44-73.
[27]刘维瑜,金春莲.多重置换扩增--一种新的全基因组扩增技术[J].国际遗传学杂志,2007,30(4):265-268.
[28]唐琴,唐秀华,孙威江.转录组学技术及其在茶树研究中的应用[J].天然产物研究与开发,2018(5):1-12.
[29]祁云霞,刘永斌,荣威恒.转录组研究新技术:RNA-Seq及其应用[J].遗传,2011,33(11):1191-1202.
[30]Wang J,Song Y.Single cell sequencing:a distinct new field[J].Clin Transl Med,2017,6(1):10.
[31]K i m K T,Lee H W,Lee H O,et al.Si ngle-cell m R NAsequencing identifies subclonal heterogeneity in anti-cancer drug responses of lung adenocarcinoma cells[J].Genome Biol,2015,16:127.
[32]Eirew P,Steif A,Khattra J,et al.Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution[J]Nature,2015,518(7539):422-426.
[33]Yu C,Yu J,Yao X,et al.Discovery of biclonal origin and a novel oncogene SLC12A5 in colon cancer by single-cell sequencing[J]Cell Res,2014,24(6):701-712.
[34]Li Y,Xu X,Song L,et al.Single-cell sequencing analysis characterizes common and cell-lineage-specific mutations in a muscle-invasive bladder cancer[J].Gigascience,2012,1(1):12.
[35]Hughes A E,Magrini V,Demeter R,et al.Clonal architecture of secondary acute myeloid leukemia defined by single-cell sequencing[J].PLoS Genet,2014,10(7):e1004462.
[36]Ramsk?ld D,Luo S,Wang Y C,et al.Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells[J].Nat Biotechnol,2012,30(8):777-782.
[37]Goetz J J,Trimarchi J M.Single-cell profiling of developing and mature retinal neurons[J].J Vis Exp,2012,62(62):e3971-e3824.
[38]Shekhar K,Lapan S W,Whitney I E,et al.Comprehensive classification of retinal bipolar neurons by single-cell transcriptomics[J].Cell,2016,166(5):1308-1323.
[39]Mahata B,Zhang X,Kolodziejczyk A A,et al.Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis[J].Cell Rep,2014,7(4):1130-1142.
[40]Shalek A K,Satija R,Adiconis X,et al.Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells[J].Nature,2013,498(7453):236-240.
[41]蔡缪荧.羔羊JIVET相关技术及影响其卵母细胞发育功能基因的研究[D].扬州:扬州大学,2015.
[42]Di R,He J,Song S,et al.Characterization and comparative profiling of ovarian microRNAs during ovine anestrus and the breeding season[J].BMC Genomics,2014,15:899.
[43]王娟红,臧明,郑毛亮,等.绵羊繁殖调控的转录组学和蛋白组学研究进展[J].中国草食动物科学,2018,38(1):49-52.
[44]兰道亮,熊显荣,陈亚冰,等.牦牛体外成熟卵母细胞差异转录组学研究[J].中国科学:生命科学,2017,47(10):1099-1112.
[45]兰道亮,熊显荣,柴志欣,等.牦牛发情期卵巢比较转录组学研究[J].畜牧兽医学报,2016,47(9):1830-1839.
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