lecture_notes:mar31
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lecture_notes:mar31 [2010/04/01 03:41] jstjohn |
lecture_notes:mar31 [2010/04/01 08:55] svasili |
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| These platforms are classified into two :\\ | These platforms are classified into two :\\ | ||
| - | 1) Sequencing by synthesis (SBS) : 454, Illumina/Solexa, Helicos, Pacbio, Charge based detection system Now Sequencing.\\ | + | * Sequencing by synthesis (SBS) : 454, Illumina/Solexa, Helicos, Pacbio, Charge based detection system Now Sequencing.\\ |
| - | 2) Sequencing by hybridization : SOLiD.\\ | + | * Sequencing by hybridization : SOLiD.\\ |
| - | __Sequencing by synthesis__\\ | + | __Sequencing by synthesis/Pyrosequencing__\\ |
| - | Involves synthesizing complementary strand of DNA to the template DNA strand to be sequenced. Four enzymes are used in this method - DNA polymerase, ATP sulfyrylase, Luciferase, and Apyrase. The light generated is captured as a signal which detects the base incorporated. The read length is approximately 400 nucleotides.\\ | + | Involves synthesizing complementary strand of DNA to the template DNA strand to be sequenced. Four enzymes are used in this method - DNA polymerase, ATP sulfyrylase, Luciferase, and Apyrase. The light generated is captured as a signal which detects the base incorporated. Currently the read length is approximately 400 nucleotides.\\ |
| + | Further description of how the 4 enzymes work in this process can be found here[[http://en.wikipedia.org/wiki/Pyrosequencing]].\\ | ||
| + | |||
| + | * 454 - Small ~20micron beads capturing DNA loaded onto PicoTiter plate along with the required enzymes for sequencing. The chemistry of the 454 sequencing can be found in detail at [[http://454.com/products-solutions/how-it-works/sequencing-chemistry.asp]].\\ | ||
| + | * Illumina/Solexa \\ | ||
| + | * Preparing DNA sample : Randomly fragment genomic DNA and ligate adapters to both ends of the fragments.\\ | ||
| + | * Immobilize DNA to surface : Bind single stranded fragments randomly to the inside surface of the flow cell channels.\\ | ||
| + | * Bridge amplification.\\ | ||
| + | * Sequence Colonies : In first chemistry cycle determine first base. Get rid of dye, otherwise chances of mis-incorporation.\\ | ||
| + | * Sequence reads over multiple chemistry cycles.\\ | ||
| + | * Read length not fixed. | ||
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| bin/ | bin/ | ||
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| bin/scripts/ | bin/scripts/ | ||
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| bin/x86_64 | bin/x86_64 | ||
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| bin/[picky_program_directories] (store programs that require the proximity of helper scripts) | bin/[picky_program_directories] (store programs that require the proximity of helper scripts) | ||
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| data/ | data/ | ||
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| programs/ (store program source and test programs before adding them to bin) | programs/ (store program source and test programs before adding them to bin) | ||
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| lib/ | lib/ | ||
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| experiments/ (store trial runs while testing out settings etc) | experiments/ (store trial runs while testing out settings etc) | ||
lecture_notes/mar31.txt · Last modified: 2010/04/01 10:44 by svasili
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