Next Generation Sequencing

Question 1

what is sanger sequencing

Answer 1

Sangers Sequencing involves: isolating the DNA, Amplification (PCR), Sequencing Reaction (ddNTPs) and Separating fragments (electrophoresis/capillary bed). sequential process

Question 2

what is next generation sequencing

Answer 2

massively parallel or deep sequencing are related terms that describe a DNA sequencing technology which has revolutionised genomic research. Using NGS an entire human genome can be sequenced within a single day

Question 3

key features of NGS

Answer 3

• It is massively parallel
• It is faster because of sequencing by synthesis (i.e. identifying bases as the synthesising DNA strand is happening).
• generates huge amounts of data

Question 4

what is meant by NGS being ‘massively parallel’

Answer 4

we’re sequencing literally billions of DNA fragments (each overlapping in sequences as more than one DNA strand is fragmented, and no DNA template is fragmented in the same position) on one instrument simultaneously.

Question 5

what are the key technologies in Illumina NGS

Answer 5

Isothermal bridge amplification – this creates the large number of single sequence spots
Sequencing by synthesis (adding a single base and then detecting it.)