鸟枪法测序原理
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Molecular weight marker every 5th lane >20 kbp
~300 bp
Contig assembly
FPC* Overlap identification by restriction pattern similarities Facilitated contig assembly
Whole Genome: 3,000 Mb
Sheared DNA: 1.0-2.0 kb
Random Reads
Sequencing Templates:
BCMHGSC
Whole Genome Shotgun Sequencing:Assembly
Low Base Quality
Single Stranded Region
Consensus Sequence Gap
Mis-Assembly (Inverted)
BCMHGSC
Whole Genome Shotgun Sequencing:Assembly
Low Base Quality
Consensus Sequence Gap
BCMHGSC
P/E ‘Celera’ Scheme:10 X coverage in three years
BCMHGSC
‘Complete an accurate, high quality sequence of the human genome by the end of 2003, …….a working draft can be completed…within the next three years…’
BCMHGSC
Consensus
Mis-Assembly (Inverted)
BCMHGSC
Shotgun Sequencing III: FINISHING
High Accuracy Sequence: < 1 error/ 10,000 bases
BCMHGSC
Whole Genome Shotgun Sequencing
‘That (draft) sequence will be of lower accuracy and contiguity…..
…will be useful for finding genes…and other features….’
BCMHGSC
‘Working Draft’ Sequence Options:
Mis-Assembly (Inverted)
BCMHGSC
Shotgun Sequencing III: FINISHING
Consensus Sequence Gap
Mis-Assembly (Inveun Sequencing III: FINISHING
- Regions not covered - Regions very densely covered - Contigs 1.0 -15 kb - # Gaps? >100,000? - Base Quality High or Low? - Mis-Assemblies? - Duplications?
Clone
A
B
C
D
E
F
G
* * *
*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.
* *
: insert fragments : vector fragments
BCMHGSC
Shotgun Sequencing I :RANDOM PHASE
Bac Clone: 100-200 kb
Sheared DNA: 1.0-2.0 kb
Random Reads
Sequencing Templates:
Low Base Quality
Single Stranded Region
Consensus Sequence Gap
Mis-Assembly (Inverted)
BCMHGSC
Shotgun Sequencing III: FINISHING
Single Stranded Region
Consensus Sequence Gap
• 0.5 -1.0 X (2 reads/kb) - ‘Skimming’
• 3.5 - 4.0 X (~9 reads/kb) -’half-shotgun’
• 6.5 - 8.0 X (~18 reads/kb) - ‘pre-finished’
• 10 X (22-24 reads/kb) - ‘deep shotgun’
BCMHGSC
Shotgun Sequencing II:ASSEMBLY
Low Base Quality
Single Stranded Region
Consensus Sequence Gap
Mis-Assembly (Inverted)
BCMHGSC
Shotgun Sequencing III: FINISHING
Regional mapping
Regional mapping
Regional mapping
Minimal tiling path selected for sequencing.
Restriction fragment fingerprinting
- BAC clones are grown in 96-well format - Hind III digest - 1% agarose