NGS Flashcards
Give an overview of NGS
- Allows millions of sequencing reactions to be carried out in parallel
- Sequencing is of clonally amplified DNA templates or single DNA molecules
What is a disadvantage of NGS and how are they combatted?
- 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
What are the three main types of sequencing chemistry?
- Sequencing by synthesis
- Sequencing by ligation
- Other developing techniques (e.g. Oxford nanopore)
Illumina sequencing used fluorescent reversible dye terminators - how does this work?
- 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
Roche’s FLX is Pyrosequencing based - how does this work?
- 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
How does ion torrent sequencing work?
- 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
Give an overview of the steps involved in NGS library prep
- 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
Give some examples of different NGS library prep methods
- Illumina TruSeq
- Illumina Nextera Rapid Capture
- Agilent Haloplex
- Agilent SureSelect
- Fluidigm
- Raindance
- Roche Nimblegen
What is the definition of read length?
Number of bases sequenced in a fragment
What is the definition of read depth?
How many times a base has been sequenced
With regards to capture efficiency what are on target and off target reads?
- on target reads capture the entire region of interest
- off target reads only capture part of the region of interest
Compare and contrast NGS and array for detection of structural variants
- 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
