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Nader talked about the chemistry behind 4 Sequencing Platforms : SOLiD Bioanalyzer, 454, Illumina/Solexa, and charge based detection system Now sequencing.
Sequencing workflow : Genomic DNA → fragment → amplification → immobilization → Sequencing.
Time required for sequencing through these platforms is :
1-2 hrs for CBD.
9 hrs for 454.
3-7 days for Illumina/Solexa.
7-10 days for SOLiD.
These platforms are classified into two :
1) Sequencing by synthesis (SBS) : 454, Illumina/Solexa, Helicos, Pacbio, Charge based detection system Now Sequencing.
2) Sequencing by hybridization : SOLiD.
Sequencing by synthesis
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.
The high level sequencing workflow for all next gen tools is as follows: Fragment Sample → Amplify Fragments → Sequence Fragments
There are two main flavors of next generation sequencing technology:
There are four technologies that were talked about today.
When a nucleotide is added to the template chain by a polymerase, a PPi is released which is converted to an ATP by one enzyme. In the presence of ATP another enzyme, luciferase, releases light. You record when the light was released (ie which nucleotide was added to the plate at that time) and also the intensity of the light. The light intensity tells you how many nucleotides were added in a row.
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 reaction.
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.
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.