Next Generation Sequencing Flashcards
What is Next Generation Sequencing (NGS)?
Method for analyzing genetic material that can sequence millions of DNA fragments at once.
What is an advantage of NGS over DNA microarray?
NGS does not reply on predefined probes.
What are the main steps of NGS?
- Library preparation
- Cluster generation
- Sequencing by synthesis
- Data analysis
What happens during NGS library preparation?
Fragmentation of sample (200-600).
Adaptors complementary to flow cell oligonucleotides are attached to ends of fragments.
Amplification (optional)
What occurs during cluster generation of NGS?
Library loaded onto flow cell and attach via adapter sequences.
Each fragment bridge amplified to form clusters (PCR)
Please explain Sequencing by Synthesis of NGS.
- Fluorescent nucleotides and DNA polymerase added to flow cell.
- Each nucleotide has a distinct fluorescent label.
- Complementary fluorescent strands synthesised.
- Sequencer captures images of the fluorescent signals from each cluster after every nucleotide addition.
Why does each fluorescent nucleotide have a reversible terminator?
To ensure only one nucleotide is incorporated into the growing DNA strand during each cycle of the sequencing process.
What happens after the sequencer captures an image of the first fluorescent nucleotide during SBS?
Cleavage solution introduced.
Reversible terminator and fluorescent tag on nucleotide are removed.
SBS process is repeated in cycles until the entire fragment is sequenced.
What are the indexes?
An index (or barcode) is a short, unique DNA sequence that is often included within or attached to the adapters.
They allow multiple samples to be sequenced together and differentiated after sequencing.
What is pair-ended sequencing?
This is when the forward and reverse strands are sequenced.
For increased accuracy.
What is demultiplexing and why/when is it done?
Demultiplexing is the process of separating fragments based on indexes.
This is done when multiple samples are pooled and share wells (polyclonal well).
What is involved in data analysis stage?
- Processing (demultiplexing, filtering and quality control)
- Read depth (coverage) calculated
- Visualisation
What is read depth/coverage?
Read depth shows how many times a specific region or nucleotide position in the genome has been sequenced.
(does not indicate which base was assigned)
(quantity of coverage)
What is an acceptable average read depth of WGS?
30 x average read depth
1500 x average read depth is good for detecting rare mutations in cancer
What is Whole Genome Sequencing (WGS)?
WGS is a specific application of NGS that involves sequencing the complete DNA sequence of an organism.
