系统生物学全册配套最完整精品课件1.ppt

1、Curriculum arrangement 1. Introduction to systems biology 2. Advanced measurement platform for -omics 3. Modeling 8(Suppl 1): S4. 28 Catastrophe theory 突变理论突变理论 Catastrophe theory, which originated with the work of the French mathematician Ren Thom in the 1960s, and became very popular due to the ef

2、forts of Christopher Zeeman in the 1970s, considers the special case where the long-run stable equilibrium can be identified with the minimum of a smooth, well-defined Lyapunov function. Small changes in certain parameters of a nonlinear system can cause equilibria to appear or disappear, or to chan

3、ge from attracting to repelling and vice versa, leading to large and sudden changes of the behaviour of the system. Ren Thom Fields Medal in 1958 Christopher Zeeman Which theory is more accurate ? Better? 29 Application: Massive genomic rearrangement acquired in a Single Catastrophic Event during Ca

4、ncer Development 在癌症发展过程中单一灾难性事件中收集到的大量基因组重在癌症发展过程中单一灾难性事件中收集到的大量基因组重 排排 FISH Profiling of TK10 Genomic Features of Chromothripsis Suggest that Most Rearrangements Occur in a Single Catastrophic Event Philip J. Stephens, et al. Cell 144, 2740, 2011 30 Hierarchical systems 分级系统分级系统 1) It is easier to

5、 analyse and (re)design large-scale systems if they are broken into smaller units. 2) The sub-system approach allows specialisation, where a sub-system is only responsible for its own task and does not require information of the objectives of the overall system. 3) Hierarchical systems allow a certa

6、in degree of fault tolerance. This is due to the fact that if a sub-system breaks down, the overall system does not necessarily stop working. Furthermore, due to module structure the failure is localised and hence easy to detect and and repair. The coordinator, however, is the weak point, because if

7、 it stops working, the overall system cannot function anymore. 4) Even evolution seems to favour two-level hierachical systems. For example in a human body the brain can be considered as being the coordinator, whereas the rest of the body forms the sub-system level. 5) In the evolution of organisati

8、ons two-level hierachical systems play a major role. Even pre- historic tribes had a tribe leader, whom was responsible for coordinating the actions of individual tribe members in order to improve the overall well-being of the tribe. Why two level hierarchical systems are so frequent? Complexity 适应性

9、 2) Parameter insensitivity;参数不敏感 3) Graceful degradation. 功能降低 34 Scale-free无标度无标度 A scale-free networks无标度网络 degree distribution follows a power law幂次定 律, that is, the fraction P(k) of nodes in the network having k connections to other nodes goes for large values of k as: p(k) k-n where n is a par

10、ameter whose value is typically in the range 2 n 100 Gb/run HiSeq 90 x106 reads/lane * 102 bp/read = 9x109 bp/lane * 16 lanes/run = 144 Gb/run 169 3. Features: uRead-lengths up to 36 bp are currently routine. uLonger reads are possible but may incur a higher error rate. Read-lengths are limited by m

11、ultiple factors多因素 that cause signal decay and dephasing移相, such as incomplete cleavage of fluorescent labels or terminating moieties. uThe dominant显性的error type is substitution代替, rather than insertions or deletions (Homopolymers均聚物 are certainly less of an issue than 454). uAverage raw error rates

12、 are on the order of 11.5%. uHigher accuracy bases with error rates of 0.1% or less can be identified through quality metrics质量度量学 associated with each base-call. 170 3. SOLiD Similar to 454 platform, in SOLiD strategy gDNA was fragmented by shotgun, and then amplified 放大放大with Emulsion PCR.微乳液微乳液PC

13、R法法 171 Clonally amplified beads are used to generate a disordered, dense array of sequencing features. Sequencing is performed with a ligase, rather than a polymerase, each sequencing cycle introduces a partially degenerate population of fluorescently labeled octamers. The population is structured

14、such that the label correlates with the identity of the central 2 bp in the octamer (the correlation with 2 bp, rather than 1 bp, is the basis of twobase encoding). After ligation and imaging in four channels, the labeled portion of the octamer (that is, zzz) is cleaved via a modified linkage betwee

15、n bases 5 and 6, leaving a free end for another cycle of ligation. Several such cycles will iteratively interrogate an evenly spaced, discontiguous set of bases. The system is then reset (by denaturation of the extended primer), and the process is repeated with a different offset (a primer set back

16、from the original position by one or several bases) such that a different set of discontiguous bases is interrogated on the next round. 172 173 174 Features: 175 4. Single Molecule Sequencer There is no clonal amplification step required. Poly-Atailed template molecules are captured by hybridization

17、 to surface- tethered poly-T oligomers to yield a disordered array of primed single molecule sequencing templates. Templates are labeled with Cy3, such that imaging can identify the subset of array coordinates where a sequencing read is expected. Each cycle consists of the polymerase-driven incorpor

18、ation of a single species of fluorescently labeled nucleotide at a subset of templates, followed by fluorescence imaging of the full array and chemical cleavage of the label. Helicos 176 Metzger M (2009) Nature Reviews Genetics 11: 31-46 177 105 to 140 Megabases per hour 35 bp average read length 17

19、8 (2009) Volume 27: 847 Helicos, 28X coverage, 84 Gb 752 CNVs 2.8M SNPs 179 Ion Torrent Single-molecule sequencing 180 Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 + - Single-molecule sequencing 181 Gupta PK. (2008) Single-mo

20、lecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 + - 182 Gupta PK. (2008) Single-molecule DNA sequencing technologies for future genomics research. Trends Biotechnol. 26:602-11 183 Pacific Biosciences Single-molecule sequencing 184 ZMW: a hole, tens of na

21、nometers in diameter, fabricated in a 100nm metal film deposited on a silicon dioxide substrate detection volume 20 zeptoliters (10-21 liters). excitationemissionemission 185 PacBio technology backgrounder: http:/ 186 When the DNA polymerase encounters the nucleotide complementary to the next base i

22、n the template, it is incorporated into the growing DNA chain. During incorporation, the enzyme holds the nucleotide in the ZMWs detection volume for tens of milliseconds, orders of magnitude longer than the average diffusing nucleotide. The system detects this as a flash of bright light because the

23、 background is very low. The polymerase advances to the next base and the process continues to repeat PacBio technology backgrounder: http:/ 187 multiple reads of the same molecule 188 Eid J et al. (2009) Molecules Real-Time DNA Sequencing from Single Polymerase Molecules. Science 323, 133 PMID: 190

24、23044 189 Does it work? 150 bp circular template 93% raw accuracy 15x coverage 99.3% accuracy 190 PacBio claims that, by 2013, the technology will be able to give a raw human genome sequence in less than 3 min, and a complete high-quality sequence in 15 min. Gupta PK. (2008) Single-molecule DNA sequ

25、encing technologies for future genomics research. Trends Biotechnol. 26:602-11 2-5 bp/sec 191 Comparison 192 Ion Torrent 193 Nature 475:348 (2011) 100 bp reads 30 Mb/run 194 195 Ion Torrent read quality 196 Applications of next-generation sequencing 197 3. Nanopore sequencing 198 199 4. Hybridizatio

26、n-based sequencing杂交杂交 Microarray DNA biochip In terms of sequencing, limitations of microarrays include the following: (i) sequences that are repetitive or subject to cross hybridization cannot easily be interrogated被询问; (ii) it remains unclear how de novo sequencing can be achieved with hybridizat

27、ion; (iii) without very careful data analysis, false positives pose an important problem, and it is not clear how to obtain the equivalent of redundant多余的 coverage that is possible with conventional常用的 and cyclic-array sequencing. It had its greatest impact in the context of genome-wide association

28、studies, which rely on array-based interrogation (that is, genotyping by hybridization) of a highly defined set of discontiguous genomic coordinates. 200 SNP 201 3.2. Transcriptomic platform Now that more and more genome sequences are being completed, new questions arise like what are the functional

29、 roles of different genes and in what cellular processes do they participate. How are genes regulated and how do genes and gene products interact. How does gene expression levels differ in various cell types and states and how is gene expression changed by various diseases or treatments. Although mR

30、NA is not the ultimate product of a gene, transcription is the first step in gene regulation and information about the transcript levels is needed for understanding gene regulatory networks. Thus, the new challenge is to identify all genes, their expression patterns and their function. Transcriptomi

31、cs or global analysis of gene expression, also called genome-wide expression profiling, is one of the tools that is used to get an understanding of genes and pathways involved in biological processes. The idea underlying this approach is called “guilt by association”, which means that genes showing

32、similarity in expression pattern may be functionally related and under the same genetic control mechanism. 202 1. Advanced platform for SB? High throughput Automatic Low cost High speed High accuracy Best gain of bioinformation in quantity and quality with unit resource or cost. 203 Genome sequence：

33、capillary array electrophoresis Gene expression profiling基因表达基因表达谱谱: biochip, RTPCR Protein sequence: separation 分分 子印章技子印章技术术;压电压电打印技打印技术术 微量点微量点样样:点接触法点接触法;喷喷墨法墨法 2.4.3. Cell chip multi microelectrode array 210 2.4.3. Cell chip chip patch clamp 2.3.4. Tissue chip 在基在基质质表面固定大量的、可表面固定大量的、可寻寻址的微小址的微小

34、组织样组织样本，用于高本，用于高 通量通量检测检测不同不同组织组织中中DNA、 、RNA和蛋白和蛋白质质等分子的等分子的变变化化 ，称，称为组织为组织芯片。芯片。 Fabrication of microfluidic chip: Method:Method: MEMSMicroelectromechanical System SiSi硅硅 or glass-based method or glass-based method Polymer聚合物聚合物-based method:PDMS SpincoatSpincoat SU-8 SU-8 Pre-exposure bakingPre-ex

35、posure baking Fix mask and wafer to holderFix mask and wafer to holder ExposureExposure Post-exposure bakePost-exposure bake Mold develop, rinse, dry and clearMold develop, rinse, dry and clear 211 Pour PDMSPour PDMS 212 Spincoat PDMS layerSpincoat PDMS layer Bake PDMSBake PDMS Cut PDMS edgeCut PDMS

36、 edge Peel off PDMSPeel off PDMS Drill holesDrill holes Channel CheckChannel Check WashWash Plasma treatmentPlasma treatment AttachmentAttachment Chip bakingChip baking Polymer-based method:PMMA 213 Soft lithography 2. Microfluidic capillary electrophoresis 微流体毛细管电泳微流体毛细管电泳 Conventional capillary el

37、ectrophoresis 常见的常见的 214 S S W B W B 2.1、进样、进样 215 S S W B W B Pinching Pinch factor: p = Ib / Is Dispense factor: d = Ibw / Is Alarie J.P. et al. Electrophoresis 2001, 22, 312-317. Electrokinetic pinching 216 S W S B W B Is + Isw + Ib + Ibw = 0 Is = Ibw Isw = Ib g = Ib / Is (gating factor) Electrok

38、inetic gating 217 CZE separation of flavin Metabolites N N N N O O OR OH OH HO OHP O OH CompoundsR RFH FAD FMN O HOOH N N N N NH2 OP O OH OP O OH -1012 2000 4000 6000 8000 10000 12000 * 3 2 1 intensity / counts time / s e.g. 218 Applications 1. DNA: genomics 2. Protein: proteomics 3. Metabolite: met

39、abolomics or metabonomics 4. Clinic diagnosis临临床床诊诊断断 5. Drug screening药药物物筛选筛选 6. Single cell analysis 7. Interaction analysis交互分析交互分析 8. Anti-bioterrorism 9. Forensic analysis法医法医鉴鉴定定 219 Quadrupole MSTOF MS 220 2.5. Bio-MS MALDI 221 ESI 500600700800900100011001200130014001500160017001800 m/z0 100

40、 % 998.2 942.7 893.2 848.6 808.3 616.2 771.5 738.0 617.2 1060.5 999.6 1131.1 1061.8 1211.8 1132.5 1137.5 1305.0 1413.7 1541.9 1696.3 160001700018000 m/z 0 100 % 16951.4 222 ESI-Q-TOF MS 223 Bragg diffraction equation： DB=BF=d sin n = 2d sin 224 2.6. X-ray crystallization 1H： ： 2.7. NMR 1924: Pauli r

41、aised the idea; 1952: Nobel Prize to Bloch(Stanford) 2. Cloned to a plasmid质粒 vector and used to transform E. coli; 3. Bacterial colony is picked and plasmid DNA isolated; 4. Each cycle sequencing reaction takes place within a microliter-scale volume, generating a ladder of ddNTP-terminated, dye-lab

42、eled products; 5. High-resolution electrophoretic separation with four channel laser induced fluorescence detection; 6. Data analysis (bioinformatics): base-calling alignment of sequence reads de novo assembly (e.g., BLAST or BLAT) genome browsing and annotation data mining 252 Advantage 1. Long rea

43、d-lengths (1000 bp) 2. High raw accuracy (99.999%) Disadvantage 1. Biological bias 偏离偏离 2. Relative high cost 0.5 $ / 1 kb 5 M$ for 1GB (10-fold coverage) The Sanger sequencing is suitable for small-scale projects in the kilobase-to- megabase range. This is a consequence of its greater granularity (

44、that is, the ability to efficiently operate at either small or large production scales) relative to the second generation technologies. 253 Dr. Mathies group at U. C. Berkeley New development: Microfluidic Capillary Array Electrophoresis 微流体毛细管阵列电泳微流体毛细管阵列电泳 One and Half generation DNA Sequencer 254

45、 2. Cyclic-array sequencing The second generation DNA sequencing: sequencing-by-synthesis Definition定定义义: The concept of cyclic- array sequencing can be defined as the sequencing of a dense稠密 array of DNA features by iterative cycles 迭 代循环of enzymatic manipulation and imaging- based data collection.

46、 General Protocol: 1. Library preparation by random fragmentation of DNA; 2. in vitro ligation of common adaptor sequences; 3. Generation of clonally无性繁殖 clustered聚集成群 amplicons扩增 (in situ polonies, emulsion PCR or bridge PCR); 4. Cyclic array sequencing; 5. Data analysis. Category: 1. 454 sequencin

47、g (Roche Applied Science, Basel); 2. Solexa (Illumina, San Diego); 3. SOLiD (Applied Biosystems, Foster City) 4. Polonator (Dover, Harvard) 5. Single Molecule Sequencer (Helicos, Cambridge) 255 1. 454 sequencing Step 1: 256 Step 2: An in vitro constructed adaptor flanked shotgun library is PCR ampli

48、fied in the context of a water-in-oil emulsion. One of the PCR primers is tethered to the surface (5- attached) of micron-scale beads that are also included in the reaction. A low template concentration results in most bead-containing compartments having either zero or one template molecule present.

49、 PCR amplicons are captured to the surface of the bead. After breaking the emulsion, beads bearing amplification products can be selectively enriched. Each clonally amplified bead will bear on its surface PCR products corresponding to amplification of a single molecule from the template library. 257

50、 Clonally amplified 28-m beads generated by emulsion PCR serve as sequencing features, are pre- incubated with Bacillus stearothermophilus (Bst) polymerase and single-stranded binding protein, and are further randomly deposited to a microfabricated array of picoliter-scale wells (with dimensions suc