基因組學(xué)與比較基因組學(xué)

1、會(huì)計(jì)學(xué)1基因組學(xué)與比較基因組學(xué)基因組學(xué)與比較基因組學(xué)第1頁(yè)/共74頁(yè)考試時(shí)間： 2009 年 1 月 12 日 下 午 2:00-4:00 考試地點(diǎn)： 三教/301(60人)、三教/304(38人)、三教/306(37人)、三教/308(37人) 第1頁(yè)/共74頁(yè)第2頁(yè)/共74頁(yè)第2頁(yè)/共74頁(yè)第3頁(yè)/共74頁(yè)History of the Human Genome Project1990 Official start of HGP with 3 billion $ and a 15 year horizon. 1999 Sanger Centre publishes chromosome 22

2、2001 Draft Genome published: Celera & Public2003 Completion (almost) of Human GenomeCelera:Craig VenterIntl. Cons:Francis Collins第3頁(yè)/共74頁(yè)第4頁(yè)/共74頁(yè)P(yáng)ublic effort- strategy:Celera - strategy:Sequencing StrategiesCeleras view of International ConsortiumInternational Consortiums view of CeleraUnfair c

3、ompetition: IC delivering the same goods but with state funding.Unfair competition: Celera delivering the same goods but can use IC data, while IC cannot use Celera data.第4頁(yè)/共74頁(yè)第5頁(yè)/共74頁(yè)第5頁(yè)/共74頁(yè)第6頁(yè)/共74頁(yè)BACBAC的構(gòu)建的構(gòu)建pBAC108L來自細(xì)菌的一個(gè)小型F質(zhì)粒，其中oriS和repE控制了質(zhì)粒的復(fù)制起始，parB和parA控制了拷貝數(shù)。100-150 Kbp insertion第6頁(yè)/共7

4、4頁(yè)第7頁(yè)/共74頁(yè)第7頁(yè)/共74頁(yè)第8頁(yè)/共74頁(yè)第8頁(yè)/共74頁(yè)第9頁(yè)/共74頁(yè)RFLP markerSSLP markersWTmut第9頁(yè)/共74頁(yè)第10頁(yè)/共74頁(yè)SNP marker第10頁(yè)/共74頁(yè)第11頁(yè)/共74頁(yè)酵母第三號(hào)染色體遺酵母第三號(hào)染色體遺傳圖（右）和物理圖傳圖（右）和物理圖（左）的比較（左）的比較 由于實(shí)驗(yàn)方法不同，不少由于實(shí)驗(yàn)方法不同，不少markers之間的遺之間的遺傳距離并不等于它們?cè)谖锢韴D上的距離。傳距離并不等于它們?cè)谖锢韴D上的距離。第11頁(yè)/共74頁(yè)第12頁(yè)/共74頁(yè)第12頁(yè)/共74頁(yè)第13頁(yè)/共74頁(yè)590bp10,000bpMate-Pair Shot

5、gun DNA Sequencing第13頁(yè)/共74頁(yè)第14頁(yè)/共74頁(yè)P(yáng)opular AssemblersTIGR Assembler (TIGR)Phrap (Wash U)Celera Assembler (Celera, TIGR)Arachne (MIT Broad)Phusion (Sanger uses Phrap)Atlas (Baylor HGSC)第14頁(yè)/共74頁(yè)第15頁(yè)/共74頁(yè)Assembly of the Individual SequencesIndividual sequencing reads are compared to eachother and whe

6、re they overlap can be assembled to create contigs第15頁(yè)/共74頁(yè)第16頁(yè)/共74頁(yè)Assembly of the Individual SequencesKeep adding individual sequencing reads to build larger and fewer contigs第16頁(yè)/共74頁(yè)第17頁(yè)/共74頁(yè)Assembly of the Individual SequencesEventually all sequencing reads merge to a single consensus sequence

7、(a large contig) for each chromosome.第17頁(yè)/共74頁(yè)第18頁(yè)/共74頁(yè)鳥槍法測(cè)序技術(shù)不能鑒別高等真核生物基因組中的重復(fù)序列鳥槍法測(cè)序技術(shù)不能鑒別高等真核生物基因組中的重復(fù)序列 第18頁(yè)/共74頁(yè)第19頁(yè)/共74頁(yè)改進(jìn)后的鳥槍法改進(jìn)后的鳥槍法(adopted by both (adopted by both IC and Celera)IC and Celera)第19頁(yè)/共74頁(yè)第20頁(yè)/共74頁(yè)目前？目前？$2/1 Mbp3 Gbp/machine/day第20頁(yè)/共74頁(yè)第21頁(yè)/共74頁(yè)DNA sequencing technologies “Cl

8、assical” Sanger dideoxy sequencing “Next Generation”, commercialized Roche 454 Pyrosequencing Solexa/Illumina cyclical base addition ABI SOLiD sequencing by ligation Single molecule (tethered DNA polymerase) Heliscope (cyclical base addition) VisiGen (real time, FRET-based)第21頁(yè)/共74頁(yè)第22頁(yè)/共74頁(yè)Illumina

9、 / Solexa Genetic Analyzer2000 Mb / runApplied Biosystems ABI 3730XL1 Mb / day Roche / 454 Genome Sequencer FLX100 Mb / runApplied BiosystemsSOLiD3000 Mb / run第22頁(yè)/共74頁(yè)第23頁(yè)/共74頁(yè)13maybe 800 bp long42第23頁(yè)/共74頁(yè)第24頁(yè)/共74頁(yè)Roche / 454 Genome Sequencer FLX100 Mb / run第24頁(yè)/共74頁(yè)第25頁(yè)/共74頁(yè)Genome sequencing in m

10、icrofabricated high-density picolitre reactors Margulies, M. Eghold, M. et al. Nature. 2005 Sep 15; 437(7057):326-7第25頁(yè)/共74頁(yè)第26頁(yè)/共74頁(yè)第26頁(yè)/共74頁(yè)第27頁(yè)/共74頁(yè)第27頁(yè)/共74頁(yè)第28頁(yè)/共74頁(yè)第28頁(yè)/共74頁(yè)第29頁(yè)/共74頁(yè)Illumina / Solexa Genetic Analyzer2000 Mb / run第29頁(yè)/共74頁(yè)第30頁(yè)/共74頁(yè)Reversible terminator-based sequencing (Solexa)第

11、30頁(yè)/共74頁(yè)第31頁(yè)/共74頁(yè)Fragment DNA and ligate adaptors第31頁(yè)/共74頁(yè)第32頁(yè)/共74頁(yè)第32頁(yè)/共74頁(yè)第33頁(yè)/共74頁(yè)第33頁(yè)/共74頁(yè)第34頁(yè)/共74頁(yè)WGS454SolexaCloningYesNoNoChemistrySangerpyrosequencing reversible terminatorsCost$ to $AccuracyConsensus 99.99%Single read 99.5%; Consensus 99.99%?AssemblyBestBetterBadGap Closure and FinishingTou

12、ghTougherPossible?第34頁(yè)/共74頁(yè)第35頁(yè)/共74頁(yè)Applied BiosystemsSOLiD3000 Mb / runOligonucleotide Ligation & Detection (SOLiD)第35頁(yè)/共74頁(yè)第36頁(yè)/共74頁(yè)SOLiD: Substrate attachment; dibase probesMake sequencing library by shearing and adapter ligationAttach DNA fragments to beads and amplify polonies in emulsionAt

13、tach beads to slide第36頁(yè)/共74頁(yè)第37頁(yè)/共74頁(yè)SOLiD: Sequencing ligation cycles第37頁(yè)/共74頁(yè)第38頁(yè)/共74頁(yè)SOLiD: Data Collection and Image Analysis第38頁(yè)/共74頁(yè)第39頁(yè)/共74頁(yè)SOLiD:Decoding thesequenceMardis 2008第39頁(yè)/共74頁(yè)第40頁(yè)/共74頁(yè)Comparison of “Next Generation”Sequencing Technologies第40頁(yè)/共74頁(yè)第41頁(yè)/共74頁(yè)第41頁(yè)/共74頁(yè)第42頁(yè)/共74頁(yè)Single M

14、olecule Sequencing Technologies: on the horizon Array of tethered DNA polymerase molecules Bound to template strand + primer Heliscope Cyclical base addition (similar to Solexa) VisiGen Real time, imaging FRET flashes Hopeful prediction: 1 Mb / sec“Next Generation” Sequencing Technologies: Rate Limi

15、ting Factors Front end: Making the sequencing library Back end: Bioinformatics to make sense of the “sequence tsunami”-essembly第42頁(yè)/共74頁(yè)第43頁(yè)/共74頁(yè)(applications) 第43頁(yè)/共74頁(yè)第44頁(yè)/共74頁(yè)Examples of Applications of “Next Generation” Sequencing TechnologiesBest for “re-sequencing”, i.e., aligning generated se

16、quence to a reference genomeNext generation DNA technologies may replace microarrays for some applicationsShendure & Ji 2008第44頁(yè)/共74頁(yè)第45頁(yè)/共74頁(yè)The “$10,000 human genome sequencing” prize To the first team that can build a device and use itto sequence: 100 human genomes within 10 days or less, Acc

17、uracy: at most 1 error per 100,000 bases, Accurate coverage of at least 98% of the genome, Recurring cost of no more than $10,000 (US) per genome. Prize: $10 million Deadline: 12:01 AM PST, October 4, 2013. Donors: X Foundation, J. Craig Venter Foundation第45頁(yè)/共74頁(yè)第46頁(yè)/共74頁(yè)Human HapMap ProjectHapMap的

18、構(gòu)建分為三個(gè)步驟：（a）在多個(gè)個(gè)體的DNA樣品中鑒定單核苷酸多態(tài)性（SNPs）；（b）將群體中頻率大于1%的那些共同遺傳的相鄰SNPs組合成單體型；（c）在單體型中找出用于識(shí)別這些單體型的標(biāo)簽SNPs。通過對(duì)圖中的三個(gè)標(biāo)簽SNPs進(jìn)行基因分型，可以確定每個(gè)個(gè)體擁有哪一個(gè)單體型。 第46頁(yè)/共74頁(yè)第47頁(yè)/共74頁(yè)SCIENCE 315:1781 (30 MARCH 2007)Metagenomes or complex samples第47頁(yè)/共74頁(yè)第48頁(yè)/共74頁(yè)第48頁(yè)/共74頁(yè)第49頁(yè)/共74頁(yè)第49頁(yè)/共74頁(yè)第50頁(yè)/共74頁(yè)small RNA identification (

19、i.e. microRNA)第50頁(yè)/共74頁(yè)第51頁(yè)/共74頁(yè)到到2006年底已完成的基因組項(xiàng)目年底已完成的基因組項(xiàng)目（/） 根據(jù)根據(jù)2007年年1月的數(shù)據(jù)，全球已啟動(dòng)月的數(shù)據(jù)，全球已啟動(dòng)2296個(gè)基因組項(xiàng)目個(gè)基因組項(xiàng)目，其中，其中607個(gè)項(xiàng)目已經(jīng)完成，已經(jīng)公開發(fā)表個(gè)項(xiàng)目已經(jīng)完成，已經(jīng)公開發(fā)表481個(gè)基因組個(gè)基因組序列，包括序列，包括403個(gè)細(xì)菌基因組，個(gè)細(xì)菌基因組，33個(gè)古細(xì)菌基因組和個(gè)古細(xì)菌基因組和45個(gè)個(gè)真核生物基因組。真核生物基因組。 其它基因組計(jì)劃其它基因組計(jì)劃第51頁(yè)/共74頁(yè)第52頁(yè)/共74頁(yè)到到2006年年12月全世界主要

20、基因組計(jì)劃的進(jìn)展情況月全世界主要基因組計(jì)劃的進(jìn)展情況 第52頁(yè)/共74頁(yè)第53頁(yè)/共74頁(yè)第53頁(yè)/共74頁(yè)第54頁(yè)/共74頁(yè)不同模式生物基因組的比較不同模式生物基因組的比較物 種基因組大小估計(jì)基因數(shù)尿殖道支原體 Mycoplasma genitalium580 Kb467肺炎支原體 Mycoplasma pneumoniae816 Kb677流感嗜血桿菌 Haemophilus influenzae1.8 Mb1709枯草芽孢桿菌 Bacillus subtilis4.2 Mb4100大腸桿菌 Escherichia coli4.6 Mb4288釀酒酵母 Saccharomyces cere

21、visiae13 Mb6275線 蟲 Caenorhabditis elegans100 Mb18891擬南芥 Arabidopsis thaliana125 Mb25498果 蠅 Drosophila melanogaster165 Mb14113人 類 Homo sapiens3 Gb約2.5萬第54頁(yè)/共74頁(yè)第55頁(yè)/共74頁(yè)物種名基因數(shù)目轉(zhuǎn)錄因子數(shù)量比例擬南芥約2938815335.9酵母約58852093.5線蟲約188916693.5果蠅約133796354.5第55頁(yè)/共74頁(yè)第56頁(yè)/共74頁(yè)Functional Categories in Eukaryotic Proteo

22、mes第56頁(yè)/共74頁(yè)第57頁(yè)/共74頁(yè)1. A key application of human genome research is to find disease genes by positional cloning2. This method involves mapping the chromosomal region containing the gene by linkage analysis in affected families3. The human genomic sequence in public databases allows rapid identific

23、ation in silico of candidate genes, followed by mutation screening of relevant candidates, aided by information on gene structure4. For a mendelian disorder, a gene search can now often be carried out in a matter of months with only a modestly sized team第57頁(yè)/共74頁(yè)第58頁(yè)/共74頁(yè)第58頁(yè)/共74頁(yè)第59頁(yè)/共74頁(yè)1. Finish

24、the human sequence2. Large-scale identification of regulatory regions3. Sequencing of additional large genomes4. Completing the catalogue of human variation5. From sequence to function第59頁(yè)/共74頁(yè)第60頁(yè)/共74頁(yè)1. Functional genomics - aims to understand how genes are regulated and what they do, largely through massively parallel studies of gene expression in a variety of tis