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lecture_notes:03-31-2010 [2010/04/01 06:08]
hyjkim
lecture_notes:03-31-2010 [2010/04/13 14:24] (current)
learithe
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 **This post was copied from [[lecture_notes:​Mar31|this original source location]]** **This post was copied from [[lecture_notes:​Mar31|this original source location]]**
  
-Nader talked about the chemistry behind 4 Sequencing Platforms : SOLiD Bioanalyzer,​ 454, Illumina/​Solexa,​ and charge ​based detection system Now sequencing.\\+Nader talked about the chemistry behind 4 Sequencing Platforms : SOLiD Bioanalyzer,​ 454, Illumina/​Solexa,​ and charge ​perturbation ​detection system Now sequencing.\\
  
 Sequencing workflow : Genomic DNA -> fragment -> amplification -> immobilization -> Sequencing.\\ ​ Sequencing workflow : Genomic DNA -> fragment -> amplification -> immobilization -> Sequencing.\\ ​
 Time required for sequencing through these platforms is :\\ Time required for sequencing through these platforms is :\\
-1-2 hrs for CBD.\\+1-2 hrs for CPD.\\
 9 hrs for 454.\\ 9 hrs for 454.\\
 3-7 days for Illumina/​Solexa.\\ 3-7 days for Illumina/​Solexa.\\
 7-10 days for SOLiD.\\ 7-10 days for SOLiD.\\
  
-These platforms ​are classified into two :\\ +The underlying technolgies for the platforms :\\ 
-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 ​Perturbation Detection ​system Now Sequencing.\\ 
-2) Sequencing by hybridization ​: SOLiD.\\+  ​* ​Sequencing by ligation ​: 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. 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]].\\
  
 +  * 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]].\\
  
-==== Alternative Notes ====+  * 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 platform can be found at [[http://​www.illumina.com/​technology/​sequencing_technology.ilmn]].\\
  
-=== High Level Overview ===+  * 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.\\
  
-The high level sequencing ​workflow ​for all next gen tools is as follows:  +  
-Fragment Sample ​-> Amplify Fragments ​-> Sequence Fragments+__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]].\\
  
-There are two main flavors of next generation sequencing technology:​ +**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.\\
-  ​Sequencing by Synthesis (SBS) +
-  ​Hybridization +
- +
-=== Examination of Individual Technologies === +
- +
-There are four technologies that were talked about today. +
-  ​Pyrosequencing (454) +
-  ​solid +
-  * Illumina/​solexa +
-  * Ion Torrent+
  
 == Pyrosequencing (SBS) == == Pyrosequencing (SBS) ==
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 After each nucleotide is added, you must wash away the nucleotides and start fresh before adding the next nucleotide and recording which positions on the plate light up in that case. After each nucleotide is added, you must wash away the nucleotides and start fresh before adding the next nucleotide and recording which positions on the plate light up in that case.
  
-There is a problem with multiple A's because ​of the modified ​nucleotides ​which are needed for this reactionFIXME+There is a problem with stretches ​of identical nucleotides. Multiple identical nucleotides will be incorporated simultaneously,​ and the sequencer must use the degree of light intensity to estimate the number of nucleotides ​incorporated; ​this calculation is prone to error. This problem, referred to as the "​homopolymeric read problem",​ is especially prounced for adenine (A).
  
-You must get the ammount ​of template on the beads just right. Must be all the same type. If too much template is present the read length is shortened (not sure why). If too few template is on the beads then the washing step will take away a significant amount of template and you won't have enough of a signal.+You must get the amount ​of template on the beads just right. Must be all the same type. If too much template is present the read length is shortened (not sure why). If too few template is on the beads then the washing step will take away a significant amount of template and you won't have enough of a signal.
  
 The packing beads that are placed around the bead which holds the template strands in the individual wells on the plate are what hold the various enzymes (other than the polymerase) needed for the reaction. The packing beads that are placed around the bead which holds the template strands in the individual wells on the plate are what hold the various enzymes (other than the polymerase) needed for the reaction.
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 == Ion Torrent (SBS) == == Ion Torrent (SBS) ==
  
-Works very similarly to pyrosequencing,​ except rather than requiring light from luciferase enzymes it takes advantage of the proton that is released when a nucleotide is added to a template. It measures this electric current. The presense ​of a current over a certain template when a specific nucleotide is added to solution tells you that that nucleotide was added to that template. The level of current at a template tells you the number of nucleotides that were added in that run.+Works very similarly to pyrosequencing,​ except rather than requiring light from luciferase enzymes it takes advantage of the proton that is released when a nucleotide is added to a template. It measures this electric current. The presence ​of a current over a certain template when a specific nucleotide is added to solution tells you that that nucleotide was added to that template. The level of current at a template tells you the number of nucleotides that were added in that run.
  
 Can sequence about 60 bases every 100 minutes. Can sequence about 60 bases every 100 minutes.
  
-Nader recommended reading the 2006 PNAS paper for background on this technology.+Nader recommended reading the [[http://​www.pnas.org/​content/​103/​17/​6466.abstract|2006 PNAS paper]] for background on this technology.
  
  
lecture_notes/03-31-2010.1270102123.txt.gz · Last modified: 2010/04/01 06:08 by hyjkim