NGS

Question 0

Describe Roche platform

Answer 0

Take amplicon pool, mix with capture beads, PCR
Load beads onto PicoTiter plate,,load enzyme beads
Pyrosequence

Question 1

Describe the illumina platform

Answer 1

Randomly fragment DNA, ligate adaptors, attach to surface, bridge amplification
Frags become ds, denature, amplify
Sequence by adding all four dNTPs and read, wash and repeat

Question 2

Describe pyrosequencing

Answer 2

Sequencing by synthesis. Chemiluminescent enzymatic reaction relies on pyrophosphate release. Visible light is generated. New dNTP added in each cycle. ATP sulfurylase converts APS (substrate) and PPi into ATP which allow luciferase to oxidate luciferin and emit light. Remaining ATP and dNTPs degraded by apyrase.

Question 3

Egs of next next generation sequencing

Answer 3

SMRT cell (single molecule real time)
Nanopore

Question 4

What are the two key advances In Sequencing

Answer 4

DNA molecules immobilized

Sequencing by synthesis