Sequencing Technologies

Question 1

Describe the process for Sanger sequencing.

Answer 1

1) Amplify DNA using PCR
2) Heat DNA to denature and separate strands
3) Add primer to 5’ end of template strand
4) Add DNA polymerase dNTPS, ddNTPs (new chains form of varying lengths until the stop ddNTP with fluorescent markers is reached) and complete PCR to get lots of strands of different lengths
5) Complete Capillary / Gel Electrophoresis to separate strands by length
6) Fluorescent markers are read off the single DNA chains to form a list of nucleotides which corresponds to the DNA sequence

Question 2

Describe the basic flow of NGS

Answer 2

1) Take tissue biopsy
2) Extract DNA from cells
3) Randomly shatter DNA
4) Library construction separates DNA templates and each one is sequenced independently

Question 3

Describe the process for NGS: Illumnia

Answer 3

1) Sample Prep - Add adaptor to end of DNA fragment
2) Cluster Generation
2a) Adaptor binds to complimentary receptor on base of plate
2b) DNA polymerase synthesizes new strand onto adapter on plate
2c) DNA is denatured and strands separate
2d) Other end of strand attaches to other adapter on plate
2e) DNA polymerase synthesizes new strand onto adapter
2f) DNA denatures
2g) reverse strands are cleaved and blocked
3) Sequencing
3a) Primers are added to the top
3b) Plate is flushed with all nucleotides
3c) Plate is excited with light and fluorescent tags on strands emit light
3d) Light is measured and nucleotide added to strand can be determined
3e) Terminator on nucleotide is removed and process is repeated

Question 4

Describe the process for NGS: Ion Torrent

Answer 4

1) DNA is cut into smaller fragments
2) 1 fragment is added onto a single bead, and copied so the entire bead is covered in the one fragment (there are millions of fragments, and millions of beads)
3)