Whole Genome Sequencing
Whole Genome Sequencing By: Qadardana kakar BS Biotechnology 7th semester
Aims/ObjectiveThe aim of my presentation is to elucidate the Whole Genome Sequencing technique and its protocol.
Sanger and his colleagues invented chain termination sequencing technique in 1970.
Fragments of 100-1000 base pair.
In 1979 shotgun sequencing technique was developed.
Large size fragments.
Overlapping of discovered fragments to detect whole genome sequences.
WGS Whole Genome Shotgun Sequencing
To sequence all genome of a particular organism.Find sequence.Find their position in entire genome. steps
Collect and isolate the DNA or genome.Break into small manageable pieces.Copy each piece many times.Read the DNA sequenceAssemble the data into a genome.
WGS Different techniques are used to cut DNA into particular size pieces.Fragments are electrophoresed through gel to find the size of fragments.
2. Ligation of fragment into a plasmid.
4. Insert plasmid into bacteria e.g. E.coli.
Bacteria with plasmid select Transformed bacteria (incubated)
5. Microtiter plate containing E.coli is heated to 95 degree Celsius.plasmids are released.6. PCR reaction amplify the plasmid.
Rolling circle amplification
7. Desired sequence amplification (Stephenson, 2003).
8. DNA attracted toward the carboxyl coated magnetic bead.In Strong ethanol solution the carboxyl coat attracts the phosphate backbone.
Cellular debris and reagents remain in solution.
Wash with ethanol. Water added to each well.
Water causes DNA to be released.
10. AssemblyMultiple read of the same genome is made.Reassembles by matching overlaps.
All Pictures are retrieved from: Whole genome sequencing/ YouTube.
Whole Genome Sequencing Advantages
To find coding non and non coding regions.
Disease susceptibility prediction.
Specie comparison and evolutionary studies.