Whole Genome Sequencing

DNA Sequencing

• Purification – isolating DNA from an organism or cells.
• Fragmentation – breaking up the genome into smaller fragments to be sequenced.
• Amplification – make more copies of our fragments.
• Sequence fragments – assigning nucleotide bases to our fragments.
• Re-assembly of fragments – put all the sequence fragments back together to create a continuous sequence.

Types of Sequencing

1. Sanger Sequencing

• Also known as the dideoxy method = dideoxynucleotides have an H on the 3’ carbon of the sugar-phosphate backbone instead of OH attached to a fluorescent tag.
• How it works:
• Amplified fragments are replicated again in the presence of the ddNTPs.
• Replication enzyme uses normal nucleotide bases and then randomly inserts a ddNTP that stops replication.
• Fluorescently labeled sequencing fragments are run through electrophoresis to separate them by size.
• The fluorescent tags are all different colors for their respective nucleotide base, need to subject gel to fluorescent filter.
• Order of sequence = shortest to longest (fragment that travelled most to fragment that travelled least).
• Most useful in sequencing single genes.

1. Whole Genome Shotgun Sequencing

• Useful in sequencing the entire genome.
• How it works:
• Isolate genome DNA and break up into overlapping fragments.
• Do this by using 2 DNA samples that have been digested by 2 different restriction enzymes.
• Clone each fragment into a plasmid vector.
• Sequence the genomic DNA fragment in each clone.
• Use computer programs to re-align sequences based on areas of overlapping.

1. Next Generation Sequencing

• Useful for multiple gene sequencing of whole genome sequencing.
• How it works:
• DNA fragments prepared by adding double stranded linkers to both ends of the fragment.
• Fragments are amplified using PCR primers that are complementary to linkers.
• Primers are then used to covalently bond the DNA fragments to a solid surface in a tight cluster.
• The fragments are analyzed using a special microscope that detects which fluorescent labeled base is added to the DNA template by polymerase.