Ion Torrent二代測(cè)序系統(tǒng)

一、國(guó)內(nèi)外研究應(yīng)用進(jìn)展

以高通量、低成本為特征的的第二代測(cè)序（next-generation sequencing, NGS）技術(shù)發(fā)展迅速，當(dāng)前風(fēng)靡測(cè)序市場(chǎng)的該類主要產(chǎn)品分別隸屬于Illumina和Life Technologies公司。我們研究所已購(gòu)置的NGS測(cè)序平臺(tái)為Life公司旗下的Ion Torrent測(cè)序平臺(tái)。Ion Torrent是基于半導(dǎo)體芯片的新一代測(cè)序技術(shù)，比其他臺(tái)式二代測(cè)序技術(shù)要更簡(jiǎn)單、更快速、性價(jià)比更高、通量靈活性更高。主要是使用一種布滿小孔的高密度半導(dǎo)體芯片，一個(gè)小孔就是一個(gè)測(cè)序反應(yīng)池。當(dāng)DNA 聚合酶把核苷酸聚合到延伸中的DNA鏈上時(shí)會(huì)釋放出一個(gè)H+，反應(yīng)池中的pH 值發(fā)生變化，位于池下的離子感受器感受到此信號(hào)，把化學(xué)信號(hào)直接轉(zhuǎn)化為數(shù)字信號(hào)，從而讀出DNA 序列。

Ion Torrent 的技術(shù)特點(diǎn)包括：1、速度快：1-2天完成所有流程，快速出結(jié)果；2、靈活性高：兼顧少量樣本和大量樣本的不同運(yùn)行需求；3、原理先進(jìn)，為首款后光學(xué)測(cè)序平臺(tái)；4、實(shí)驗(yàn)和結(jié)果分析流程高度自動(dòng)化；5、唯一同時(shí)滿足長(zhǎng)讀長(zhǎng)和高通量的平臺(tái)。

目前國(guó)內(nèi)外Ion Torrent測(cè)序系統(tǒng)的醫(yī)學(xué)相關(guān)應(yīng)用主要集中在：1、病原微生物檢測(cè)，包括微生物群落鑒定、微生物變異體鑒定、微生物耐藥性檢測(cè)。相對(duì)于傳統(tǒng)的培養(yǎng)分離法，依賴測(cè)序技術(shù)的宏基因組分析能十分精確地揭示微生物種類和遺傳的多樣性，為微生物群落結(jié)構(gòu)分析提供直觀而全面的信息，對(duì)了解和監(jiān)測(cè)治療由多種微生物引起的感染性疾病有重要意義。而隨著自然選擇作用和生物體的不斷進(jìn)化，越來越多的微生物變異體悄然出現(xiàn)，當(dāng)這些新的變異體引發(fā)的感染性疾病肆虐時(shí)，人們往往措手不及。因此，如何快速鑒定這些新的變異體有重要意義。Ion Torrent PGM快速明確了引發(fā)德國(guó)2011年腸出血性大腸桿菌（EHEC）暴發(fā)疫情的病原體及其進(jìn)化關(guān)系和耐藥基因，對(duì)遏制疫情起到了重要作用。而對(duì)已知的微生物耐藥基因進(jìn)行測(cè)序，能夠更直觀、準(zhǔn)確、細(xì)致地在分子水平顯示耐藥基因的變異，對(duì)科研開發(fā)和臨床用藥有重要的指導(dǎo)意義。2、腫瘤檢測(cè)。隨著轉(zhuǎn)化醫(yī)學(xué)的發(fā)展，大量具有臨床意義的腫瘤相關(guān)基因?qū)?huì)被發(fā)現(xiàn)，因此，大范圍地進(jìn)行基因檢測(cè)也是腫瘤防治的有效手段。Ion Torrent創(chuàng)建了Ion AmpliSeq腫瘤測(cè)序平臺(tái)，將腫瘤研究轉(zhuǎn)化為一管式分析，能產(chǎn)生最多480個(gè)擴(kuò)增子，覆蓋46個(gè)腫瘤基因的相關(guān)區(qū)域，讓低頻突變能夠得到準(zhǔn)確經(jīng)濟(jì)的檢測(cè)。隨著Ion Torrent測(cè)序平臺(tái)的進(jìn)一步擴(kuò)大，例如乳腺癌高危家族中屬于抑制基因的BRCA基因突變的檢測(cè)費(fèi)用很有可能降低到200 美元以下，使Ion Torrent 技術(shù)能夠更廣泛地應(yīng)用于臨床重要基因突變的篩查。3、遺傳病的檢測(cè)。異常的有遺傳性的家族性疾病或者由地域環(huán)境引起的高發(fā)病甚至某類人群的高發(fā)疾病都可通過高通量的NGS技術(shù)實(shí)現(xiàn)對(duì)其全基因組信息的讀取而探究發(fā)病的遺傳基礎(chǔ)，從而指導(dǎo)臨床治療方案的選擇。此外，在當(dāng)前生殖醫(yī)學(xué)高速發(fā)展的今天，尤其在試管嬰兒大量出生的時(shí)代背景下，如何做好優(yōu)質(zhì)胚胎的選擇，即胚胎植入前的基因鑒別的任務(wù)就依靠于NGS的優(yōu)勢(shì)發(fā)揮，結(jié)合了當(dāng)前發(fā)展較為成熟的單細(xì)胞測(cè)序技術(shù)，可以從基因組學(xué)上實(shí)現(xiàn)很好的胚胎選擇。Ion Torrent PGM 針對(duì)遺傳病設(shè)計(jì)了AmpliSeq遺傳病測(cè)序平臺(tái)，3 個(gè)反應(yīng)池包含超過10 000 對(duì)引物，可以擴(kuò)增與700多種遺傳病相關(guān)的328種基因的外顯子，包括神經(jīng)肌肉類、心腦血管類、生長(zhǎng)發(fā)育類和遺傳代謝類疾病。4、其他應(yīng)用。除了普通的基因序列的讀取辨別外，NGS也可對(duì)一系列的RNA進(jìn)行測(cè)序，建立某些表達(dá)譜，還可以根據(jù)RNA序列反向驗(yàn)證某些基因編碼序列的遺傳背景。此外，對(duì)于那些基于大范圍DNA甲基化所產(chǎn)生的環(huán)境相關(guān)疾病，更適合采用NGS技術(shù)進(jìn)行DNA甲基化的測(cè)序工作，以此解釋某些疾病發(fā)生的深層機(jī)制。

二、該平臺(tái)在研究所的作用和意義

北京市肝病研究所，為一所市屬公益性科研院所，旨在進(jìn)一步拓寬肝病的研究領(lǐng)域，加快肝病防治工作的進(jìn)程，減少肝臟疾病的發(fā)生，利用佑安醫(yī)院的特色病種，將臨床治療與基礎(chǔ)研究緊密結(jié)合起來，創(chuàng)建一支創(chuàng)新、發(fā)展的科研團(tuán)隊(duì)。HBV－慢性肝炎－肝癌是至今原因不明，是遺傳、免疫背景很強(qiáng)的疾病。研究HBV感染者個(gè)體化轉(zhuǎn)歸差異的分子免疫學(xué)和肝細(xì)胞分子網(wǎng)絡(luò)差異改變，尋求肝硬化和肝癌發(fā)生的分子機(jī)制對(duì)于防控肝癌的發(fā)生，降低肝癌發(fā)病率和死亡率都具有重要的意義。

在基因組測(cè)序技術(shù)快速進(jìn)步以及生物信息與大數(shù)據(jù)科學(xué)的交叉應(yīng)用的大環(huán)境下，精準(zhǔn)醫(yī)療應(yīng)運(yùn)而生。精準(zhǔn)醫(yī)療時(shí)代下的高通量基因測(cè)序發(fā)展正酣，尤其在遺傳性出生缺陷的診斷和預(yù)防，腫瘤發(fā)生、發(fā)展與治療的分子機(jī)制，各類重大遺傳病的遺傳分析等領(lǐng)域，尚有無限廣闊的空間有待臨床醫(yī)務(wù)工作者和科研人員配合開拓。

在我國(guó)每年近28 萬人死于肝硬化、肝癌等肝臟相關(guān)疾病，中國(guó)新發(fā)和死亡肝癌患者數(shù)量占全球一半以上，其中多數(shù)是因病毒性肝炎而致。能夠?yàn)榘┌Y患者提供準(zhǔn)確的基因分析，給臨床醫(yī)生提供靶向藥物和臨床試驗(yàn)信息，最后給出最精確的個(gè)體化治療方案是每個(gè)醫(yī)學(xué)工作者的夢(mèng)想。研究所Ion Torrent二代測(cè)序系統(tǒng)平臺(tái)的建立可以實(shí)現(xiàn)綜合分析全基因組、全轉(zhuǎn)錄組數(shù)據(jù)，發(fā)現(xiàn)肝炎、肝硬化和肝癌相關(guān)基因組、轉(zhuǎn)錄組水平相關(guān)信息，檢測(cè)不同類型患者的轉(zhuǎn)錄水平和基因突變差異，同時(shí)也為人才梯隊(duì)建設(shè)和學(xué)科骨干人才的培養(yǎng)，創(chuàng)造了條件。測(cè)序技術(shù)的高度發(fā)展會(huì)使全基因組序列讀取更加便利和經(jīng)濟(jì)，個(gè)體化臨床應(yīng)用也會(huì)隨之更加廣闊和深入，由此將實(shí)現(xiàn)一個(gè)生命科學(xué)與醫(yī)療診治交互作用的全新前景，從而實(shí)現(xiàn)肝臟病的精準(zhǔn)醫(yī)療。

三、診斷和科研用途

（一）分子診斷

1、腫瘤個(gè)體化分子診斷

1.1 EGFR-TKIs藥物與EGFR基因突變

1.2 克唑替尼與ALK基因突變

1.3 靶向藥物與KRAS基因突變

1.4 靶向藥物與BRAF基因突變

1.5 胃腸道間質(zhì)瘤及格列衛(wèi)藥物與C-KIT基因、PDGFRA基因

1.6 P53基因突變與腫瘤

1.7 骨髓增殖性疾病與JAK2基因突變

1.8 家族性乳腺/卵巢癌與BRCA1/2基因突變

2、藥物代謝相關(guān)分子診斷

2.1 伊立替康與UGT1A1基因多態(tài)性

2.2 華法林藥物與CYP2C9、VKORC1和CYP4F2基因分型

3、遺傳病分子診斷

3.1遺傳性心律失常疾病

3.2單基因遺傳病系列：地中海貧血、遺傳性耳聾、苯丙酮尿癥、血友病

4、血液系統(tǒng)疾病分子診斷

4.1 HLA高分辨率基因分型

4.2 白血病

5、婦嬰生育保健分子診斷：主要為無創(chuàng)產(chǎn)前DNA檢測(cè)（NIPT）

6、感染性疾病分子診斷

6.1感染性疾病基因分型：微生物16S rRNA鑒定

6.2 藥物耐藥相關(guān)基因：乙型肝炎病毒耐藥基因突變

（二）科研用途

1、靶向測(cè)序：

相對(duì)于全基因組測(cè)序，靶向測(cè)序是一種快速、高性價(jià)比方法。Ion AmpliSeq? 技術(shù)對(duì)這一應(yīng)用進(jìn)行了轉(zhuǎn)化，使得研究人員可使用僅僅10 ng DNA即可快速簡(jiǎn)單地對(duì)數(shù)千靶標(biāo)進(jìn)行擴(kuò)增。

2、轉(zhuǎn)錄組測(cè)序：

轉(zhuǎn)錄組測(cè)序，也稱為RNA測(cè)序(RNA-Seq)，可提供有關(guān)基因組組織和調(diào)控方式的基礎(chǔ)信息。RNA測(cè)序依賴于二代測(cè)序 (NGS) 方法和技術(shù)。

3、異倍性及CNV分析：

拷貝數(shù)變異(CNV)分析是一種可用于異倍性等染色體畸變分析的二代測(cè)序方法。Ion PGM?系統(tǒng)二代測(cè)序提供了一種簡(jiǎn)單、快速的技術(shù)，能夠可靠地獲得易分析的數(shù)據(jù)。

4、小RNA及miRNA測(cè)序：

結(jié)合尖端的Ambion? RNA技術(shù)與易用的Ion Torrent?二代測(cè)序方法，任意實(shí)驗(yàn)室均可使用小RNA測(cè)序來了解發(fā)現(xiàn)更多信息。

5、病毒基因分型：

Ion PGM?系統(tǒng)結(jié)合PathAmp? FluA試劑及用于Torrent Suite?軟件的病原體分析插件，可以使研究人員在不到一天內(nèi)完成甲型流感分型，獲得的高精度序列數(shù)據(jù)，從而對(duì)流感追溯研究樣品進(jìn)行更有效的分析。

6、外顯子組測(cè)序：

外顯子組測(cè)序是一種靶向測(cè)序方法，可對(duì)基因組上與疾病發(fā)生相關(guān)的外顯子組區(qū)域進(jìn)行特異性研究。Life Technologies為提供了靈活、簡(jiǎn)單、價(jià)格實(shí)惠的Ion Proton?系統(tǒng)外顯子組測(cè)序方案。

7、微生物測(cè)序：

隨著Ion PGM? 系統(tǒng)的通量越來越高、精確度越來越準(zhǔn)確、讀段越來越長(zhǎng)，其逐漸被用來對(duì)特定疾病類型的追溯樣品進(jìn)行分析、監(jiān)測(cè)，研究其發(fā)作方式，并進(jìn)一步確定其病原學(xué)信息。

8、測(cè)序法基因分型：

人的基因組遺傳變異存在多種形式，從較大的、完整結(jié)構(gòu)的染色體變化到單核酸多態(tài)性(SNPs)都屬于這種變異類型。Life Technologies?提供了一系列產(chǎn)品來對(duì)遺傳變異及基因組表達(dá)譜進(jìn)行分析。

9、從頭測(cè)序：

Ion Torrent?半導(dǎo)體測(cè)序技術(shù)為微生物研究提供了革命性的從頭測(cè)序方法，是一套可在一天內(nèi)給出精確結(jié)果的簡(jiǎn)單、低成本系統(tǒng)。

10、細(xì)菌基因分型：

Ion PGM?系統(tǒng)可對(duì)成百上千的基因進(jìn)行分型研究，快速、經(jīng)濟(jì)地給出全基因組序列信息，從而對(duì)于單一菌株獲得更高的分辨能力和更精確的分型信息。

四、最近幾年發(fā)表的與此相關(guān)的文章

1、Ion circulating tumor cells (CTCs) and cell-free DNA (cfDNA) Publications:

[1]Kelley RK, Magbanua MJ, Butler TM, et al. Circulating tumor cells in hepatocellular carcinoma: a pilot study of detection,  enumeration, and next-generation sequencing in cases and controls. BMC Cancer. 2015;15: 206.

[2]Frenel JS, Carreira S, Goodall J, et al. Serial Next Generation Sequencing of Circulating Cell Free DNA Evaluating Tumour  Clone Response To Molecularly Targeted Drug Administration. Clinical Cancer Research. 2015.

[3]Rothe F, Laes JF, Lambrechts D, et al. Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer. Annals of Oncology. 2014;25: 1959-65.

[4]Couraud S, Vaca-Paniagua F, Villar S, et al. Noninvasive diagnosis of actionable mutations by deep sequencing of circulating free DNA in lung cancer from never-smokers: a proof-of-concept study from BioCAST/IFCT-1002. Clinical Cancer Research. 2014;20: 4613-24.

[5]Carreira S, Romanel A, Goodall J, et al. Tumor clone dynamics in lethal prostate cancer. Science Translational Medicine. 2014;6: 125r-254r.

[6]Kukita Y, Uchida J, Oba S, et al. Quantitative identification of mutant alleles derived from lung cancer in plasma  cell-free DNA via anomaly detection using deep sequencing data. PLoS One. 2013;8: e81468.2、Ion Exome Sequencing Publications

2、Ion Exome Sequencing Publications:

[1]Oliveira J, Negrao L, Fineza I, et al. New splicing mutation in the choline kinase beta (CHKB) gene causing a muscular dystrophy detected by whole-exome sequencing.Journal of Human Genetics. 2015;60: 305-12.

[2]Wakil SM, Monies DM, Abouelhoda M, et al. Association of a mutation in LACC1 with a monogenic form of systemic juvenile idiopathic arthritis. Arthritis Rheumatol. 2015;67: 288-95.

[3]Severson PL, Vrba L, Stampfer MR, Futscher BW.Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells.Mutat Res Genet Toxicol Environ Mutagen. 2014;775-776: 48-54.

[4]Monies DM, Al-Hindi HN, Al-Muhaizea MA, et al. Clinical and pathological heterogeneity of a congenital disorder of glycosylation manifesting as a myasthenic/myopathic syndrome. NeuromusculDisord. 2014;24: 353-59.

[5]Motoike IN, Matsumoto M, Danjoh I, et al. Validation of multiple single nucleotide variation calls by additional exome analysis with a semiconductor sequencer to supplement data of whole-genome sequencing of a human population. BMC Genomics. 2014;15: 673.

[6]Boland JF, Chung CC, Roberson D, et al. The new sequencer on the block: comparison of Life Technology's Proton sequencer  to an IlluminaHiSeq for whole-exome sequencing. Human Genetics. 2013;132: 1153-63.

[7]Chong IY, Cunningham D, Barber LJ, et al. The genomic landscape of oesophagogastricjunctional adenocarcinoma.Journal of Pathology. 2013;231: 301-10.

3、Ion RNA-seq Publications:

[1]Narayan A, Bommakanti A, Patel AA. High-throughput RNA profiling via up-front sample parallelization. Nature Methods. 2015;12: 343-46.

[2]Smyth LJ, McKay GJ, Maxwell AP, McKnight AJ. DNA hypermethylation and DNA hypomethylation is present at different loci in chronic kidney disease. Epigenetics. 2014;9: 366-76.

[3]Thanh NM, Jung H, Lyons RE, et al. A transcriptomic analysis of striped catfish (Pangasianodonhypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery. Comp BiochemPhysiol Part D Genomics Proteomics. 2014;10: 52-63.

[4]Cheng L, Sharples RA, Scicluna BJ, Hill AF. Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood. J Extracell Vesicles. 2014;3.

[5]Nozawa M, Fukuda N, Ikeo K, Gojobori T. Tissue- and stage-dependent dosage compensation on the neo-X chromosome in Drosophila pseudoobscura. Molecular Biology and Evolution. 2014;31: 614-24.

[6]Li S, Tighe SW, Nicolet CM, et al. Multi-platform assessment of transcriptome profiling using RNA-seq in the ABRF next-generation sequencing study. Nature Biotechnology. 2014;32: 915-25.

4、Ion Targeted RNA Sequencing Publications:

[1]Lenz N, Schindler T, Kagina BM, et al. Antiviral Innate Immune Activation in HIV-Infected Adults Negatively Affects H1/IC31-Induced Vaccine-Specific Memory CD4+ T Cells. Clinical and Vaccine Immunology. 2015;22: 688-96.

[2]Poole A, Urbanek C, Eng C, et al. Dissecting childhood asthma with nasal transcriptomics distinguishes subphenotypes of disease. J Allergy ClinImmunol. 2014;133: 670-78.

5、Ion Viral Sequencing Publications:

[1]Yi X, Zou J, Xu J, et al. Development and validation of a new HPV genotyping assay based on next-generation sequencing. American Journal of Clinical Pathology. 2014;141: 796-804.

[2]Briese T, Mishra N, Jain K, et al. Middle East respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from  dromedary camels in Saudi Arabia. MBio. 2014;5: e1114-46.

[3]Gibson RM, Meyer AM, Winner D, et al. Sensitive deep-sequencing-based HIV-1 genotyping assay to simultaneously determine susceptibility to protease, reverse transcriptase, integrase, and maturation inhibitors, as well as HIV-1 coreceptor tropism. Antimicrob Agents Chemother. 2014;58: 2167-85.

6、Ion Targeted DNA Sequencing Publications:

[1]Hovelson DH, McDaniel AS, Cani AK, et al. Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumors. Neoplasia. 2015;17: 385-99.

[2]Wang T, Zhan X, Bu CH, et al. Real-time resolution of point mutations that cause phenovariance in mice. ProcNatlAcadSci U S A. 2015;112: E440-49.

[3]Warrick JI, Hovelson DH, Amin A, et al. Tumor evolution and progression in multifocal and paired non-invasive/invasive urothelial carcinoma. VirchowsArchiv. 2015;466: 297-311.

[4]Baquero-Montoya C, Gil-Rodriguez MC, Braunholz D, et al. Somatic mosaicism in a Cornelia de Lange syndrome patient with NIPBL mutation identified by different next generation sequencing approaches. Clinical Genetics. 2014;86: 595-97.

[5]Beck J, Pittman A, Adamson G, et al. Validation of next-generation sequencing technologies in genetic diagnosis of dementia. Neurobiology of Aging. 2014;35: 261-65.

[6]Bell CC, Magor GW, Gillinder KR, Perkins AC. A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations  using next-generation sequencing. BMC Genomics. 2014;15: 1002.

[7]Ezgu F, Ciftci B, Topcu B, et al. Diagnosis of glycine encephalopathy in a pediatric patient by detection of a GLDC mutation during initial next generation DNA sequencing. Metabolic Brain Disease. 2014;29: 211-13.

[8]Niba ET, Tran VK, Tuan-Pham LA, et al. Validation of ambiguous MLPA results by targeted next-generation sequencing discloses a nonsense mutation in the DMD gene. ClinicaChimicaActa. 2014;436: 155-59.

[9]Rechsteiner M, Muller R, Reineke T, et al. Modelling of a genetically diverse evolution of Systemic Mastocytosis with Chronic Myelomonocytic Leukemia (SM-CMML) by Next Generation Sequencing. ExpHematolOncol. 2014;3: 18.

[10]Woyach JA, Furman RR, Liu TM, et al. Resistance mechanisms for the Bruton's tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370: 2286-94.

[11]Kukita Y, Uchida J, Oba S, et al. Quantitative identification of mutant alleles derived from lung cancer in plasma  cell-free DNA via anomaly detection using deep sequencing data. PLoS One. 2013;8: e81468.

7、Ion Metagenomic Publications:

[1]Mondav R, Woodcroft BJ, Kim EH, et al. Discovery of a novel methanogen prevalent in thawing permafrost. Nature Communications. 2014;5: 3212.

[2]Ly M, Abeles SR, Boehm TK, et al. Altered oral viral ecology in association with periodontal disease. MBio. 2014;5: e1114-33.

8、Ion Genotyping by Sequencing Publications:

[1]Mascher M, Wu S, Amand PS, Stein N, Poland J. Application of genotyping-by-sequencing on semiconductor sequencing platforms: a  comparison of genetic and reference-based marker ordering in barley. PLoS One. 2013;8: e76925.

9、Ion Viral Typing Publications:

[1]Zhang Y, Mao H, Yan J, et al. Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China. Virus Research. 2014;189: 158-64.

[2]Zhou B, Lin X, Wang W, et al. Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics. Journal of Clinical Microbiology. 2014;52: 1330-37.

10、Ion NIPT and Aneuploidy Detection Publications:

[1]Liao C, Yin AH, Peng CF, et al. Noninvasive prenatal diagnosis of common aneuploidies by semiconductor sequencing. ProcNatlAcadSci U S A. 2014;111: 7415-20.

五、研究所可提供的臨床和科研服務(wù)項(xiàng)目

（一）臨床服務(wù)項(xiàng)目

1、腫瘤個(gè)體化分子診斷：主要為肝癌相關(guān)

2、婦嬰生育保健分子診斷：針對(duì)佑安醫(yī)院產(chǎn)科，可進(jìn)行無創(chuàng)產(chǎn)前DNA檢測(cè)（NIPT）

3、感染性疾病分子診斷：如HBV/HIV藥物耐藥相關(guān)基因突變檢測(cè)

（二）科研用途

1、靶向測(cè)序

2、轉(zhuǎn)錄組測(cè)序

3、外顯子組測(cè)序

4、微生物測(cè)序

5、從頭測(cè)序

Fluidigm單細(xì)胞自動(dòng)制備及單細(xì)胞測(cè)序表達(dá)譜系統(tǒng)

一、國(guó)內(nèi)外研究應(yīng)用進(jìn)展

單細(xì)胞測(cè)序技術(shù)可謂是科技發(fā)展史上的一大創(chuàng)舉。細(xì)胞是生物學(xué)的基本單位，研究人員正努力嘗試將它們進(jìn)行單個(gè)分離、研究和比較。一個(gè)細(xì)胞里的DNA或RNA僅僅處在皮克（picograms）級(jí)的水平，如此少的量遠(yuǎn)遠(yuǎn)達(dá)不到現(xiàn)有測(cè)序儀的最低上樣需求。目前生物學(xué)研究中的基因組測(cè)序多是提取大量細(xì)胞中的遺傳物質(zhì)后進(jìn)行的，忽略了細(xì)胞間的差異，而單細(xì)胞測(cè)序可以避免這種情況。因此科學(xué)家們必須先對(duì)單細(xì)胞內(nèi)的微量核酸分子進(jìn)行擴(kuò)增，而且必須保證盡可能少地出現(xiàn)技術(shù)誤差，以便開展后續(xù)的測(cè)序及其他研究。一些科學(xué)家認(rèn)為，通過研究單個(gè)、完整細(xì)胞內(nèi)的遺傳物質(zhì)，有朝一日可以理解細(xì)胞特別是腦細(xì)胞的工作機(jī)制。而了解癌細(xì)胞如何在腫瘤內(nèi)變化以及每個(gè)細(xì)胞內(nèi)“定居”著多少版本的基因，相關(guān)技術(shù)有望用于癌癥診斷。

Fluidigm微流體系統(tǒng)通過將巨量的流體元件集成于單一微流體芯片，克服了諸多傳統(tǒng)實(shí)驗(yàn)室系統(tǒng)的局限性。Fluidigm技術(shù)使其客戶在利用微量試劑和樣品的同時(shí)，能夠采用小于一個(gè)細(xì)胞容量的樣品執(zhí)行和測(cè)量數(shù)以千計(jì)的尖端復(fù)雜生物化學(xué)反應(yīng)。同樣，對(duì)于下一代 DNA測(cè)序，F(xiàn)luidigm系統(tǒng)能夠以較低的成本迅速準(zhǔn)備多個(gè)樣本。Fluidigm單細(xì)胞自動(dòng)制備及單細(xì)胞測(cè)序表達(dá)譜系統(tǒng)由C1?系統(tǒng)、Juno?系統(tǒng)和BioMark? HD系統(tǒng)組成。C1單細(xì)胞全自動(dòng)制備系統(tǒng)是全世界最先推出的全自動(dòng)單細(xì)胞分離和制備系統(tǒng)。C1系統(tǒng)為每次運(yùn)行處理96個(gè)單細(xì)胞提供了簡(jiǎn)單輕松、高度重復(fù)的流程，而手工操作所需的時(shí)間極少。它前所未有地讓分離細(xì)胞、提取、逆轉(zhuǎn)錄和擴(kuò)增過程實(shí)現(xiàn)全面自動(dòng)化，使細(xì)胞活性的檢測(cè)和分析成為可能，并減少了多平臺(tái)技術(shù)錯(cuò)誤所引起的可變性。Juno系統(tǒng)為全新SNP基因分型平臺(tái)，在不到3小時(shí)的時(shí)間內(nèi)處理并完成低至2.5ng/ul的DNA樣本的SNP基因分型（如組織，口腔拭子，血，石蠟包埋組織和多倍體生物），有效突破了基因分型領(lǐng)域目前對(duì)高濃度、高質(zhì)量DNA要求的瓶頸。BioMark? HD高通量基因分析系統(tǒng)是集成流體通路（Integrated Fluidic Circuit，IFC）技術(shù)、實(shí)時(shí)定量PCR技術(shù)及強(qiáng)大的基因分析軟件相結(jié)合的技術(shù)平臺(tái)。BioMark HD系統(tǒng)由BioMark?實(shí)時(shí)PCR系統(tǒng)（整合了高性能計(jì)算機(jī)）、IFC微液流芯片（耗材）、IFC Controller（將生物樣品、反應(yīng)試劑導(dǎo)入到IFC微液流芯片中）和基因分析軟件四部分構(gòu)成。IFC微液流芯片有2種：Dynamic Array（48.48動(dòng)態(tài)芯片和96.96動(dòng)態(tài)芯片）和Digital Array（12.765數(shù)碼芯片和48.770數(shù)碼芯片），應(yīng)用于不同的基因分析中：拷貝數(shù)變化分析、遺傳突變檢測(cè)、基因分型、基因定量、單細(xì)胞基因表達(dá)等。

單細(xì)胞研究的核心問題其實(shí)是：為什么要進(jìn)行單細(xì)胞研究？這主要是因?yàn)槿绻麑⒊汕先f個(gè)細(xì)胞混在一起進(jìn)行研究，就會(huì)模糊我們對(duì)大腦、血液系統(tǒng)、免疫系統(tǒng)，及其組成這些系統(tǒng)的細(xì)胞之間異質(zhì)性（heterogeneity）的認(rèn)識(shí)。當(dāng)研究深入到單細(xì)胞層面時(shí)，就可能會(huì)失去對(duì)整個(gè)系統(tǒng)的把控，但是如果能夠從整個(gè)系統(tǒng)中挑選多個(gè)不同的單細(xì)胞進(jìn)行研究，則可以重建出整個(gè)系統(tǒng)，而且這種重建過程能夠提供更多更有價(jià)值的信息。比較罕見的細(xì)胞、異質(zhì)性的樣本、與遺傳嵌合或突變相關(guān)的表型、不能人工培養(yǎng)的微生物，這些都是單細(xì)胞測(cè)序技術(shù)能夠一展所長(zhǎng)的研究平臺(tái)。使用單細(xì)胞測(cè)序技術(shù)能夠發(fā)現(xiàn)克隆突變（clonal mutation）、隱藏的細(xì)胞類型，或者在大塊組織樣品研究工作中被“稀釋”或平均掉的轉(zhuǎn)錄特征。目前單細(xì)胞研究主要集中在遺傳學(xué)、神經(jīng)科學(xué)、腫瘤及微生物生態(tài)學(xué)等領(lǐng)域，如單細(xì)胞生物的單細(xì)胞測(cè)序、人類單體型（human haplotypes）研究、體細(xì)胞突變研究等。對(duì)單細(xì)胞的基因表達(dá)情況進(jìn)行檢測(cè)和分析非常有助于我們了解細(xì)胞的行為，以及明確都有哪些細(xì)胞參與了組織發(fā)育、成熟和病變的過程。單細(xì)胞RNA測(cè)序的實(shí)現(xiàn)使得步入一個(gè)單細(xì)胞轉(zhuǎn)錄組學(xué)時(shí)代，該研究方向會(huì)對(duì)生物學(xué)和醫(yī)學(xué)產(chǎn)生深刻的影響。單細(xì)胞RNA測(cè)序技術(shù)尤其適用于對(duì)體內(nèi)的腫瘤細(xì)胞進(jìn)行分析，因?yàn)獒槍?duì)一堆轉(zhuǎn)化細(xì)胞、間質(zhì)細(xì)胞和其它浸潤(rùn)細(xì)胞單獨(dú)提取轉(zhuǎn)錄產(chǎn)物進(jìn)行分析，可以了解各種轉(zhuǎn)錄產(chǎn)物的豐度和亞型信息。對(duì)離散的腫瘤組織和健康組織進(jìn)行單細(xì)胞轉(zhuǎn)錄組分析還可以精確地確定與轉(zhuǎn)化狀態(tài)相關(guān)的、不同的mRNA亞型。轉(zhuǎn)錄組上的差異也有助于我們認(rèn)識(shí)腫瘤的進(jìn)展情況，更好地認(rèn)識(shí)CTC細(xì)胞的異質(zhì)性問題，幫助大家更好地認(rèn)識(shí)CTC細(xì)胞進(jìn)入血液循環(huán)系統(tǒng)時(shí)的基因表達(dá)情況。技術(shù)上的新進(jìn)展已經(jīng)讓單細(xì)胞基因組測(cè)序技術(shù)（single-cell genome sequencing）逐漸成為了一項(xiàng)主流的檢測(cè)手段，該領(lǐng)域的研究工作已經(jīng)初步揭示出細(xì)胞之間在基因組結(jié)構(gòu)（genetic architecture）與遺傳變異性（genetic variability）方面的差異，這也反映出基因組并非一成不變的天然本質(zhì)。

通常，將具有同一表型的細(xì)胞看作是一個(gè)具有特定功能的整體，并將其稱作組織或者器官。不過對(duì)單個(gè)細(xì)胞進(jìn)行深度DNA和RNA測(cè)序會(huì)發(fā)現(xiàn)，各種各樣的細(xì)胞狀態(tài)構(gòu)成了一個(gè)復(fù)雜的生態(tài)系統(tǒng)，這樣一個(gè)復(fù)雜的系統(tǒng)才形成了組織和器官的整體功能。繼續(xù)發(fā)展高信息度、實(shí)時(shí)的、多模單細(xì)胞檢測(cè)技術(shù)將有助于真正認(rèn)識(shí)處于微環(huán)境系統(tǒng)下單個(gè)細(xì)胞的功能。對(duì)單細(xì)胞的DNA和RNA進(jìn)行深度測(cè)序就能夠以前所未有的更高的分辨率，更全面地掌握細(xì)胞的功能。對(duì)細(xì)胞狀態(tài)的這種特異性識(shí)別能力有助于我們更好地了解細(xì)胞的正常功能和異常情況。

二、該平臺(tái)在研究所的作用和意義

北京市肝病研究所，為一所市屬公益性科研院所，旨在進(jìn)一步拓寬肝病的研究領(lǐng)域，加快肝病防治工作的進(jìn)程，減少肝臟疾病的發(fā)生，利用佑安醫(yī)院的特色病種，將臨床治療與基礎(chǔ)研究緊密結(jié)合起來，創(chuàng)建一支創(chuàng)新、發(fā)展的科研團(tuán)隊(duì)。HBV－慢性肝炎－肝癌是至今原因不明，是遺傳、免疫背景很強(qiáng)的疾病。研究HBV感染者個(gè)體化轉(zhuǎn)歸差異的分子免疫學(xué)和肝細(xì)胞分子網(wǎng)絡(luò)差異改變，尋求肝硬化和肝癌發(fā)生的分子機(jī)制對(duì)于防控肝癌的發(fā)生，降低肝癌發(fā)病率和死亡率都具有重要的意義。

2013年，單細(xì)胞測(cè)序技術(shù)（single-cell sequencing）榮膺《自然-方法》年度技術(shù)。單細(xì)胞測(cè)序技術(shù)有助于剖析細(xì)胞的異質(zhì)性。它可以揭示腫瘤細(xì)胞基因組中發(fā)生的突變及結(jié)構(gòu)性變異，而這些突變和變異往往有著極高的突變率。有了這些信息，就可以描述腫瘤細(xì)胞的克隆結(jié)構(gòu)，并追蹤疾病的進(jìn)展及擴(kuò)散范圍。

在我國(guó)每年近28 萬人死于肝硬化、肝癌等肝臟相關(guān)疾病，中國(guó)新發(fā)和死亡肝癌患者數(shù)量占全球一半以上，其中多數(shù)是因病毒性肝炎而致。研究所Fluidigm單細(xì)胞自動(dòng)制備及單細(xì)胞測(cè)序表達(dá)譜系統(tǒng)的建立可以實(shí)現(xiàn)單細(xì)胞制備分離、單細(xì)胞基因組及轉(zhuǎn)錄組測(cè)序。高通量的病變組織單細(xì)胞分析能夠同時(shí)檢測(cè)細(xì)胞的組成變化（通過細(xì)胞聚類分析手段）和相應(yīng)的基因表達(dá)變化。我們可以對(duì)健康組織和病變組織里特定的細(xì)胞進(jìn)行比較，發(fā)現(xiàn)與疾病相關(guān)的特異性基因表達(dá)改變情況。對(duì)肝癌患者單個(gè)CTC細(xì)胞進(jìn)行轉(zhuǎn)錄組學(xué)分析是一種無創(chuàng)檢測(cè)手段，可幫助臨床醫(yī)生們選擇合適的抗癌藥物和治療方案，隨時(shí)監(jiān)測(cè)病情的進(jìn)展情況和療效，還可以根據(jù) CTC細(xì)胞上的分子標(biāo)志物確定將來的靶向治療方案。我們可以以新的視角看待HBV－慢性肝炎－肝癌的發(fā)生機(jī)制。

三、診斷和科研用途

1、單細(xì)胞全基因組測(cè)序

基于單個(gè)細(xì)胞水平上采取高質(zhì)量的全基因組擴(kuò)增與測(cè)序相結(jié)合的一項(xiàng)新技術(shù)，該技術(shù)目前主要應(yīng)用于腫瘤發(fā)生機(jī)制及胚胎發(fā)育研究。由于腫瘤細(xì)胞之間具有異質(zhì)性，采用該項(xiàng)技術(shù)不需培養(yǎng)細(xì)胞，可最真實(shí)的獲得單克隆癌細(xì)胞的具體突變來源及精準(zhǔn)的突變頻率，以及區(qū)分癌癥發(fā)生、發(fā)展、演化過程中的主動(dòng)與被動(dòng)突變等。

2、單細(xì)胞轉(zhuǎn)錄組測(cè)序

利用高質(zhì)量的基因組擴(kuò)增技術(shù)，對(duì)單個(gè)細(xì)胞水平上的mRNA進(jìn)行測(cè)序分析的技術(shù)。該技術(shù)可以從單細(xì)胞水平解釋基因表達(dá)異常及RNA編輯現(xiàn)象在癌癥的發(fā)生、發(fā)展、演化過程中的作用。

3、Single-cell targeted gene expression

4、Single-cell MicroRNA

四、最近幾年發(fā)表的與此相關(guān)的文章

1、Analysis Methods Publications:

[1]Trapnell, C. et al. “The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells.” NatureBiotechnology 32(4) (2014): 381–6.*

2、Cancer Research Publications:

[1]Benavides-Garcia, R. et al. “Granulocyte colony-stimulating factor prevents loss of spermatogenesis after sterilizing busulfanchemotherapy.” Fertility and sterility 103(1) (2014): 270–280.e8.*

[2]Gawad, C. et al. “Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics.” Proceedings of theNational Academy of Sciences of the United States of America 111(50) (2014): 1,7947–52.*

[3]Azizi, E. et al. “The promise of single cell omics for onco-therapy.” Journal of molecular and genetic medicine: an international journal ofbiomedical research 8 (2014): 3.*

[4]Ennen, M. et al. “Single-cell gene expression signatures reveal melanoma cell heterogeneity.” Oncogene (2014): doi: 10.1038*

[5]Landau, D. et al. “Locally disordered methylation forms the basis of intratumormethylome variation in chronic lymphocyticleukemia.”Cancer Cell 26(6) (2014): 813–25.*

[6]Papaemmanuil, E. et al. “RAG-mediated recombination is the predominant driver ofoncogenic rearrangement in ETV6-RUNX1 acutelymphoblastic leukemia.” Nature Genetics 46(2) (2014): 116–25.

3、Cardiovascular Research Publications:

[1]Chini, V. et al. “Micro-RNAs and next generation sequencing: new perspectives in heart failure.” Clinicachimicaacta (2014):doi: 10.1016/j.cca.2014.11.020.*

[2]Fu, J. et al. “Direct reprogramming of human fibroblasts toward a cardiomyocyte-like state.” Stem Cell Reports 1(3) (2013): 235–47.*

4、Circulating Tumor Cells Publications:

[1]Ozkumur, E. et al. “Inertial focusing for tumor antigen– dependent and –independent sorting of rare circulating tumor cells.” ScienceTranslational Medicine 5(179) (2013): 179ra47.

[2]Powell, A.A. et al. “Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines.”PLoS ONE 7 (2012): (5):e33788.

[3]Helzer, K.T. et al. “Circulating tumor cells are transcriptionally similar to the primary tumor in a murine prostate model.” Cancer Research69(19) (2009): 7,860–6.

5、Embryonic Stem Cells and DevelopmentPublications:

[1]Brunskill, E.W. et al. “Single cell dissection of early kidney development: multilineage priming.” Development 141(15) (2014): 3,093–101.*

[2]Behrens, A. et al. “Sox7 is regulated by Etv2 during cardiovascular development.” Stem Cells and Development 23(17) (2014): 2,004–13.*

[3]Duscher, D. et al. “Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells.” ScientificReports 4 (2014): 7144.

[4]Turner, D.A. et al. “Wnt/β-catenin and FGF signalling direct the specification and maintenance of a neuromesodermal axial progenitor inensembles of mouse embryonic stem cells.” Development 141(22) (2014): 4,243–53.

[5]Li, N. et al. “Single-cell analysis of proxy reporter allele-marked epithelial cells establishes intestinal stem cell hierarchy.” Stem CellReports 3(5) (2014): 876–91.

6、Gene Expression ProfilingPublications:

[1]Dorrell, C. et al. “The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar.” Stem Cell Reports2 (2014): 275–83.

[2]Bennett, R. et a. “Laser microdissection of the alveolar duct enables single-cell genomic analysis.” Frontiers in Oncology 4 (2014): 260.*

[3]Shalek, A.K. et al. “Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells.” Nature 498(7453) (2013):236–40.

[4]Buczacki, S.J. et al. “Intestinal label-retaining cells are secretory precursors expressing Lgr5.” Nature 495(7439) (2013): 65–9.

[5]McDavid, A. et al. “Data exploration, quality control and testing in single-cell qPCR-based gene expression experiments.” Bioinformatics29(4) (2013): 461–7.

7、Hematopoietic Stem Cells and ProgenitorsPublications:

[1]Grover, A. et al. “Erythropoietin guides multipotent hematopoietic progenitor cells toward an erythroid fate.” Journal of ExperimentalMedicine 211(2) (2014): 181–8.*

[2]Becher, B. et al. “High-dimensional analysis of the murine myeloid cell system.” Nature Immunology 12 (2014): 1,181–9.**

[3]Miyawaki, K. et al. “CD41 marks the initial myelo-erythroid lineage specification in adult mouse hematopoiesis: Redefinition of murinecommon myeloid progenitor.” Stem Cells (2014): doi: 10.1002/stem.1906.

8、Immunity/Infectious DiseasePublications:

[1]Wang, J. et al. “RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection.” Proceedings of theNational Academy of Sciences of the United States of America 111(36) (2014):13,157–62.*

[2]Shalek A.K. et al. “Single-Cell RNA-seq reveals dynamic paracrine control or cellular variation.” Nature 509(7,505) (2014): 363–9.*

[3]Mahata, B. et al. “Single-cell RNA sequencing reveals T helper cells synthesizing steriodsde novo to contribute to immunehomeostasis.” Cell Reports 7(4) (2014): 1,130–42.*

[4]Strauss-Albee, D.M. et al. “Coordinated regulation of NK receptor expression in the maturing human immune system.” Journal ofImmunology 193(10) (2014): 4,871–9.**

[5]Swadling, L. et al. “A human vaccine strategy based on chimpanzee adenoviral and MVA vectors that primes, boosts, and sustainsfunctional HCV-specific T cell memory.” Science Translational Medicine 6(261) (2014): 261ra153.**

9、Induced Pluripotent Stem Cells (IPS)Publications:

[1]Dominguez, A. et al. “Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosomeaneuploidies.” Scientific Reports 4 (2014): 6,432.

[2]Chanda, S. et al. “Generation of Induced Neuronal Cells by the Single Reprogramming Factor ASCL1.” Stem Cell Reports 3(2) (2014):282–96.

[3]Burridge, P.W. et al. “Chemically defined generation of human cardiomyocytes.” Nature Methods 11(8) (2014): 855–60.

10、MASS CYTOMETRYPublications:

[1]Edgar, L.J. et al. “Identification of Hypoxic Cells Using an Organotellurium Tag Compatible with Mass Cytometry.” Science TranslationalMedicine 53(43) (2014): 11,473–7.**

[2]Giesen, C. et al. “Highly multiplexed imaging of tumor tissues with subcellular resolution by mass cytometry.” Nature Methods 4 (2014):417–22.**

[3]Leipold, M.D. et al. “Mass cytometry: protocol for daily tuning and running cell samples on a CyTOF mass cytometer.” Journal ofVisualized Experiments 69 (2012): e4398.**

11、Neural ResearchPublications:

[1]Romanov, R. et a. “A secretagogin locus of the mammalian hypothalamus controls stress hormone release.” The EMBO Journal 34(1)(2014): 36–54.*

[2]Victor, M. et al. “Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts.” Neuron 84(2) (2014):311–23.

[3]Park, J. et al. “Identifying functional gene regulatory network phenotypes underlying single cell transcriptional variability.” Progress inbiophysics and molecular biology S0079–6107(14) (2014): 00183–7.

12、New MethodsPublications:

[1]Yao, Y. et al. “CyTOF supports efficient detection of immune cell subsets from small samples.” Journal of Immunological Methods 145(2014): 1–5.**

[2]Behbehani, G. et al. “Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining.”Cytometry Part A 85(12) (2014): 1,011–9.**

[3]O’Neill, K. et al. “Enhanced flowType/RchyOptimyx: A bioconductor pipeline for discovery in high-dimensional cytometry data.”Bioinformatics 30(9) (2014): 1,329–30.**

[4]Amir el, A.D. et al. “viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.”Nature Biotechnology 31(6) (2013): 545–52.**

13、RNA SEQPublications:

[1]Tang, X. et al. “The eSNV-detect: a computational system to identify expressed single nucleotide variants from transcriptomesequencing data.” Nucleic Acids Research 42(22) (2014): e172.*

[2]Pollen, A.A. et al. “Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways indeveloping cerebral cortex.” Nature Biotechnology 32(10) (2014): 1,053–8.*

[3]Saliba A.E. et al. “Single-cell RNA-seq: advances and futurechallenges.” Nucleic Acids Research 42(14) (2014): 8,845–60.*

[4]Treutlein, B. et al. “Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.” Nature 509(7,500) (2014):371–5.*

[5]Islam, S. et al. “Quantitative single-cell RNA-seq with unique molecular identifiers.” Nature Methods 11(2) (2013): 163–6.*

[6]Wu, A.R. et al. “Quantitative assessment of single-cell RNA-sequencing methods.” Nature Methods 11(1) (2013): 41–6.*

[7]Brennecke, P. et al. “Accounting for technical noise in single-cell RNA-Seq experiments.” Nature Methods 10(11) (2013): 1,093–5.*

五、研究所可提供的臨床和科研服務(wù)項(xiàng)目

1、單細(xì)胞全基因組測(cè)序

2、單細(xì)胞轉(zhuǎn)錄組測(cè)序

3、Single-cell targeted gene expression

聯(lián)系人：魏飛力、歐陽雅博

聯(lián)系電話：010-63057109/010-83997424/010-83997425

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