NGS

Question 1

Give an overview of NGS

Answer 1

• Allows millions of sequencing reactions to be carried out in parallel
• Sequencing is of clonally amplified DNA templates or single DNA molecules

Question 2

What is a disadvantage of NGS and how are they combatted?

Answer 2

• Read lengths shorter than Sanger and Error rate for individual NGS reads is greater

Combatted:

• NGS Accuracy achieved by sequencing given region multiple times generating a consensus sequence
• to assemble, align and analyse data requires adequate number of NGS reads

Question 3

What are the three main types of sequencing chemistry?

Answer 3

1. Sequencing by synthesis
2. Sequencing by ligation
3. Other developing techniques (e.g. Oxford nanopore)

Question 4

Illumina sequencing used fluorescent reversible dye terminators - how does this work?

Answer 4

• only one nucleotide incorporated at each cycle
• after incorporation the array is imaged, terminator moiety cleaved and fluorescent label is cleaved off and removed
• fresh nucleotides added and the cycle is repeated

Question 5

Roche’s FLX is Pyrosequencing based - how does this work?

Answer 5

• beads into microtitre plates which are arrayed into a picotiter plate (fused silica capillary structure)
• this holds a single bead in each of several hundred thousand single wells, providing a fixed location at which each sequencing reaction can be monitored by Pyro
• Pyrophosphate is converted to ATP. ATP drives the luciferase-mediated conversion of Luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP
• Luminescence is transmitted through the fibre optic plate and recorded in a charge-coupled device camera

Question 6

How does ion torrent sequencing work?

Answer 6

• sequencing by synthesis occurs by sequential addition of unmodified nucleotides
• unlike Pyro, seq occurs by direct electrical detection (hydrogen ions released as a result of the synthesis reaction)
• this is translated into a voltage signal and subsequently into a base call
• no cameras, light sources or scanners involved - only a digital voltage change is used for detection

Question 7

Give an overview of the steps involved in NGS library prep

Answer 7

• input DNA
• fragmentation (nebulisation, shearing or enzymatic digestion) to generate fragments in size range of 150-600bp (platform dependent)
• Fragments have terminal overhangs that require blunt end repair and phosphorylation
• Ligation of adaptors
• Purification of repaired fragments by spin column or magnetic beads
• Hybridisation to sequences complementary to adaptor sequences (emulsion PCR or bridge amplification)
• Amplification

Question 8

Give some examples of different NGS library prep methods

Answer 8

• Illumina TruSeq
• Illumina Nextera Rapid Capture
• Agilent Haloplex
• Agilent SureSelect
• Fluidigm
• Raindance
• Roche Nimblegen

Question 9

What is the definition of read length?

Answer 9

Number of bases sequenced in a fragment

Question 10

What is the definition of read depth?

Answer 10

How many times a base has been sequenced

Question 11

With regards to capture efficiency what are on target and off target reads?

Answer 11

• on target reads capture the entire region of interest

- off target reads only capture part of the region of interest

Question 12

Compare and contrast NGS and array for detection of structural variants

Answer 12

• Array doesn’t detect balanced SVs
• NGS can detect both balanced and unbalanced SVs
• Much higher resolution with NGS than array
• Short reads of NGS limit ability to map SVs
• Solution is to use paired end reads/mate pair library