Next Generation Sequencing

Question 1

What are the basic steps in next-gen sequencing?

Answer 1

DNA library construction, cluster generation, sequencing-by-synthesis, data analysis

Question 2

How do you construct a DNA library?

Answer 2

Chop the sample up into small pieces -> repair ends -> add adenine overhangs -> ligate adaptors

Question 3

How do you generate clusters?

Answer 3

Hybridise DNA library fragments to flowcell, bridge amplification

Question 4

How do you do sequencing-by-synthesis?

Answer 4

Incorporating single nucleotides, washing the flowcell, imaging the four bases and then cleaving the terminator chemical group and dyeing with an enzyme

Question 5

What is a DNA library?

Answer 5

A collection of random DNA fragments of a specific sample used for further study

Question 6

Next gen sequencing vs sanger sequencing

Answer 6

NGS produces a digital readout - sanger produces an analogue readout, sanger is a one sequence read and NGS Is a consensus sequence of many reads

Question 7

How do you do whole exome sequencing?

Answer 7

Target enrichment, then you capture the regions of interest with baits

Question 8

What size is the exome in whole exome sequencing?

Answer 8

50 Mb

Question 9

How do you carry out exome data analysis?

Answer 9

Sequence read alignment with the reference genome, target coverage reporting, variant calling, variant annotation

Question 10

When would you use exome sequencing

Answer 10

Identifying the variant shared by a group of affected individuals