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lecture_notes:mar31 [2010/04/01 01:48]
svasili
lecture_notes:mar31 [2010/04/01 03:44] (current)
svasili
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 7-10 days for SOLiD.\\ 7-10 days for SOLiD.\\
  
-These platforms ​are classified into two :\\+The underlying technolgies for the platforms :\\
   * 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.\\
-  * Sequencing by hybridization ​: SOLiD.\\+  * Sequencing by ligation ​: SOLiD.\\
  
 __Sequencing by synthesis/​Pyrosequencing__\\ __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. Currently 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. Incorporation of homopolymer A is found to be problematic.\\
 Further description of how the 4 enzymes work in this process can be found here[[http://​en.wikipedia.org/​wiki/​Pyrosequencing]].\\ 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]].\\   * 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 \\   * Illumina/​Solexa \\
-    * Randomly fragment genomic DNA and ligate adapters to both ends of the fragments+    * 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 platform can be found at [[http://​www.illumina.com/​technology/​sequencing_technology.ilmn]].\\ 
 + 
 +  * 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. No problem is detected so far with the incorporation of homopolymer A.\\ 
 + 
 +  
 +__Sequencing by Ligation__\\ 
 +ABI SOLiD sequencing technology follows sequencing by ligation. In first ligation cycle, possible 1024 oligonucleotide probes compete for incorporation,​ followed by dephosphorylation,​ visualization,​ and cleavage. Same procedure is followed for the next ligation cycles. Every 5th round of cycle, the process is reset. In the next cycle, primer length is reduced by one base (n-1) to get overlap, and again in the subsequent cycles the primer length is n-2, n-3 and so on to determine the DNA sequence due to overlap. For further reading follow [[http://​sequencing.soe.ucsc.edu/​node/​20]].\\ 
 + 
 +**Mate-Pairing** - The chances of damage to DNA increases with time. Hence, there is a high possibility of not getting the accurate sequence data from the other end of DNA. To avoid this, the Mate-Pairing process involves the determination of sequence of DNA from the end towards the beginning and then from the beginning towards the end.\\
   ​   ​
  
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-Also we meet in the science library this Friday.+Also we meet in the science library ​(instructions room near periodicals) ​this Friday.
  
  
  
    
lecture_notes/mar31.1270111716.txt.gz · Last modified: 2010/04/01 01:48 by svasili