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Whole Genome
Shotgun Sequencing
By-Gouthaman P P
Introduction
Sequencing entire genome
Evolutionary studies
Metabolic pathway/ Disease pathway
Disease susceptibility prediction based on variation
History
Shotgun sequencing has been developed for sequencing of large
fragments of DNA in 1979.
DNA is broken up randomly into numerous small segments, which
are sequenced using the chain termination method and then short
reads have been produced.
Shotgun sequencing was the initiative for full genome sequencing.
In this strategy, the DNA is first shredded into smaller fragments
which can be sequenced individually. The sequences of these
fragments are then reassembled into their original order, based on
overlaps, ultimately yielding the complete sequence.
Whole genome shotgun sequencing
Whole genome shotgun sequencing
Whole genome shotgun sequencing
Whole genome shotgun sequencing
Whole genome shotgun sequencing
Whole genome shotgun sequencing
Procedure
Genomic DNA is fragmented by sonification or hydrodynamic
shearing
All sticky-end fragments are blunt ended with T4 DNA
polymerase and exonuclease activity
T4 polynucleotide kinase is added so that 5' ends are
phosphorylated
Fragments separated into either small (~1kb), medium (~8kb) and
large (~40kb) fragments
A library is created per each size in plasmids and transformed
into E. coli cells
Vector DNA is purified from each library and amplified
Each DNA strand is sequenced
Computer program called a ’base caller’ filters out poor calls
The ’assembler’ finds overlapping segments and generates long
successive continuous stretches of nucleotides, called contigs.
Whole genome shotgun sequencing
Whole Genome Shotgun
• Huge percentage of human genome is repeated non-coding
regions that are difficult to sequence
• Gene Myers proposed to break the whole genome into pieces of
2000bp, 10000bp and 50000bp respectively
• Unique segments were sequenced while using repeats as markers
to rebuild the sequence
• The result was compiled and full stretches of Genome was
generated using computational power
THANK YOU

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Whole genome shotgun sequencing

  • 2. Introduction Sequencing entire genome Evolutionary studies Metabolic pathway/ Disease pathway Disease susceptibility prediction based on variation
  • 3. History Shotgun sequencing has been developed for sequencing of large fragments of DNA in 1979. DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method and then short reads have been produced. Shotgun sequencing was the initiative for full genome sequencing.
  • 4. In this strategy, the DNA is first shredded into smaller fragments which can be sequenced individually. The sequences of these fragments are then reassembled into their original order, based on overlaps, ultimately yielding the complete sequence.
  • 11. Procedure Genomic DNA is fragmented by sonification or hydrodynamic shearing All sticky-end fragments are blunt ended with T4 DNA polymerase and exonuclease activity T4 polynucleotide kinase is added so that 5' ends are phosphorylated Fragments separated into either small (~1kb), medium (~8kb) and large (~40kb) fragments
  • 12. A library is created per each size in plasmids and transformed into E. coli cells Vector DNA is purified from each library and amplified Each DNA strand is sequenced Computer program called a ’base caller’ filters out poor calls The ’assembler’ finds overlapping segments and generates long successive continuous stretches of nucleotides, called contigs.
  • 14. Whole Genome Shotgun • Huge percentage of human genome is repeated non-coding regions that are difficult to sequence • Gene Myers proposed to break the whole genome into pieces of 2000bp, 10000bp and 50000bp respectively • Unique segments were sequenced while using repeats as markers to rebuild the sequence • The result was compiled and full stretches of Genome was generated using computational power