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lecture_notes:mar31 [2010/04/01 03:41] jstjohn |
lecture_notes:mar31 [2010/04/01 09:31] 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]].\\ | ||
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+ | * 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]].\\ | ||
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+ | * 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.\\ | ||
+ | * The chemistry of Illumina sequencing plaform can be found at [[http://www.illumina.com/technology/sequencing_technology.ilmn]].\\ | ||
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+ | * Now Sequencing Platform (CMOS based sequencing platform) - Sequencing based on detection of charge when a base is incorporated. Link to the article published by Nader in this respect is [[http://www.pnas.org/content/103/17/6466]]. On an average this sequencing technology incorporates upto 2.5 nucleotides in 2 minutes and in 60 minutes upto 100 bases.\\ | ||
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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) | ||