北京大学细胞生物学.pptxVIP

第十一章 基因组学与比较基因组学;基础分子生物学期末安排;基因组计划;History of the Human Genome Project;Public effort- strategy:;Public effort- strategy:基因组DNA大片段文库的构建 ;BAC的构建 pBAC108L来自细菌的一个小型F质粒，其中oriS和repE控制了质粒的复制起始，parB和parA控制了拷贝数。;遗传图谱(Genetic Map)vs 物理图谱 (Physical Map);DNA遗传标记 (DNA marker) ;RFLP marker;SNP marker;酵母第三号染色体遗传图（右）和物理图（左）的比较 ;鸟枪法序列测定技术;DNA target sample;大规模DNA序列拼接 ;;;;鸟枪法测序技术不能鉴别高等真核生物基因组中的重复序列 ;改进后的鸟枪法(adopted by both IC and Celera);高通量DNA序列分析技术 ;DNA sequencing technologies ? “Classical” – 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); ;;Roche / 454 : Genome Sequencer FLX;Pyrosequencing---454 Sequencing;A section of Pyrosequencing reads;Illumina / Solexa: Genetic Analyzer;Reversible terminator-based sequencing (Solexa);Fragment DNA and ligate adaptors; Comparison; Real Time Sequencing by Ligation Emulsion PCR and Beads on slides 85 million reads / run Up to 35 bases/read 3 Gb / run dual fluorescent labels 8 individual channels / flowcell 2 flowcells / run ;SOLiD: Substrate attachment; dibase probes Make sequencing library by shearing and adapter ligation Attach DNA fragments to beads and amplify polonies in emulsion Attach beads to slide;SOLiD: Sequencing ligation cycles;SOLiD: Data Collection and Image Analysis;SOLiD: Decoding the sequence;Comparison of “Next Generation” Sequencing Technologies;Applications Genetic Analyzer;Single Molecule 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;What do we sequence?;Examples of Applications of “Next Generation” Sequencing Technologies Best for “re-sequencing”, i.e., aligning generated