3. WHOLE GENOME SEQUENCING
•All genome has unique genetic code or
genome that is composed of bases ( A,T,C,G).
•WGS ia an invitro technique which is used to
determine the nucleotide sequence of the
genome of that organism .
•Whole-genome sequencing is a process that
determines the DNA sequence of an entire
genome.
4. •In 1977, Sanger also used his method to sequence
the first ever complete genome: the one of the
bacteriophage PhiX174 (virus that infects E. coli).
•Also in 1977, Maxam and Gilbert introduced a
method for DNA sequencing that was based on
chemical modification of DNA.
•The WGS is invented by the J. craig venter and H.
smith in 1995.
History
5. The whole genome sequencing comprises of
various steps to sequence the whole genome .
1. Firstly the DNA is cutted into several pieces
using different techniques .
2. Then the fregmented pieces of DNA is ligated
into the plasmid.
3. then the plasmid contaning the desired
fragment of DNA is inserted into the Ecoli.
4. Micro titer plates containing the ecoli is heated
to 95 degree C for the separation of the
plasmid and release of the DNA.
5. Now the DNA fragments are amplified
Workflow of WGS
6. 6. The amplified DNA attracted towards the carboxyl coated magnatic beads, These beads
help in the purification and sepration of the dna for the preparation of DNA library
7. The DNA library is prepared and the small DNA tags or barcodes are added to the DNA
contigs which will help in the identification of the DNA .
8. The refered genome is added to the sequencer according to which the DNA assambles and
sequenced
9. The DNA sequences is done.
7.
8. •The short gun sequencing is used for the sequencing of long DNA strands
•Dna is broken up randomly into the numerous small segments, which are
sequenced by the chain termination method.
•Multiple overlapping reds for the target DNA are obtained by performing
the several rounds of this fragmentation and sequencing.
•Computer program then use overlapping ends of the different reads to
assemble them into continuous sequence.
Short gun sequencing
9.
10. These analysis method involved can be
time consuming and computationally
heavy often requiring complex and
expensive infrastructures.
Limitation
11. The method preferred by the human genome project is the hierarchical shotgun sequencing
method.
•Also known as the clone by clone sequencing
•The map based method top down sequencing
Clone by clone sequencing
12. •The genome is split into larger fragments ( 50-200kb) using restricted cutting enzymes that
contains a known marker.
•These fragments are closed in bacteria (e.coli) using BACs where they replicated and stored.
•The BACs inserts are isolated and the whole genome is mapped by markers.
•The fragments contained in these clones have different ends.
•Each BACs fragments is fragmented randomly into smaller pieces and these fragments are
individually sequenced using the automated Sanger sequencing.
•These sequences are aligned so that identical sequences are overlapping.
•The assembly of the genome is done on the basis of prior knowledge of the marker used to
localize sequenced fragments to their genomic location.
Steps
