鸟枪法测序原理.pptVIP

Regional mapping Regional mapping Regional mapping Contig assembly * Minimal tiling path selected for sequencing. * * 20 kbp ~300 bp Molecular weight marker every 5th lane Restriction fragment fingerprinting - BAC clones are grown in 96-well format - Hind III digest - 1% agarose Clone A B C D E F G FPC* Overlap identification by restriction pattern similarities Facilitated contig assembly *Sanger Centre C. Soderlund, I Longden and R. Mott * * * * * * All restriction fragments within a clone selected for the tiling path must be verified by their presence in overlapping clones. : vector fragments : insert fragments BCM- HGSC Shotgun Sequencing I :RANDOM PHASE Bac Clone: 100-200 kb Sheared DNA: 1.0-2.0 kb Sequencing Templates: Random Reads BCM- HGSC Shotgun Sequencing II:ASSEMBLY Consensus Sequence Gap Low Base Quality Single Stranded Region Mis-Assembly (Inverted) BCM- HGSC Consensus Sequence Gap Low Base Quality Single Stranded Region Mis-Assembly (Inverted) BCM- HGSC Shotgun Sequencing III: FINISHING Consensus Sequence Gap Single Stranded Region Mis-Assembly (Inverted) BCM- HGSC Shotgun Sequencing III: FINISHING Consensus Sequence Gap Mis-Assembly (Inverted) BCM- HGSC Shotgun Sequencing III: FINISHING Consensus Mis-Assembly (Inverted) BCM- HGSC Shotgun Sequencing III: FINISHING BCM- HGSC Shotgun Sequencing III: FINISHING High Accuracy Sequence: 1 error/ 10,000 bases Whole Genome Shotgun Sequencing Whole Genome: 3,000 Mb Sheared DNA: 1.0-2.0 kb Sequencing Templates: Random Reads BCM- HGSC Whole Genome Shotgun Sequencing:Assembly Consensus Sequence Gap Low Base Quality Single Stranded Region Mis-Assembly (Inverted) BCM- HGSC Whole Genome Shotgun Sequencing:Assembly Consensus Sequence Gap Low Base Quality BCM- HGSC P/E ‘Celera’ Scheme:10 X coverage in three years - Contigs 1.0 -15 kb - # Gaps? 100,000? - Base Quality High or Low? BCM- HGSC - Mis-Assemblies? - Duplications? - Regions not covered - Regions very densely covered ‘