Block D Lecture 4: High-Throughput Sequencing and Epilepsy Flashcards
What are the 7 key steps in a whole genome sequencing (WGS) project?
- Isolate genomic DNA from patient
- Fragment DNA (shotgun approach)
- Prepare genomic library (add adaptors)
- Sequence
- Quality control - Read trimming and quality filtering
- Reference mapping
- Potential consequences for patient
(Slide 3)
What occurs in the library generation step (step 1) of illumina sequencing chemistry?
Genomic DNA is fragmented (either physically or by an enzyme) and then adaptors are ligated onto the DNA fragments to immobilise DNA onto flow cell
(Slide 8)
What occurs in the cluster generatrion step (step 2) of illumina sequencing chemistry?
The flow cell is coated in complementary adapters and the DNA library is diluted so that fragments are evenly distributed across the flow cell.
Then, bridge PCR amplifies each fragment at that location, forming a cluster
(Slide 9)
What occurs in the sequencing by synthesis step (step 3) of illumina sequencing chemistry?
Fluorescent bases are inserted and the flow cell image is recorded in real time.
The colour of the cluster indicates the base being incorporated at that moment
(Slide 11)
Is flueorescent chain termination reversible or irreversible?
Reversible
(Slide 11)
Why can adaptors have barcodes / index sequences?
So that multiple samples can be mixed together (total sequencing capacity of an instrument is often much greater than coverage required)
(Slide 13)
What does Pacbio SMRT sequencing produce?
Longer reads with a lower accuracy
(Slide 17)
What does the Pacbio SMRT sequencing method immobilise?
The polymerase
(Slide 18)
What score does every base in a read have associated with it?
A quality score
(Slide 23)
What tends to be the trend in quality scores of bases as you get to the end of a read?
They decrease
(Slide 23)
What 4 things does the FastQ raw data format include?
What sequencing platform and conditions were used
The sequence
Spacer row
Quality string encoded representation of the Phred score
(Slide 25)
What is a seizure?
A paroxysmal behavioural spell generally caused by an excessive disorderly discharge of cortical nerve cells
(Slide 27)
What does paroxysmal mean?
A sudden recurrence or intensification of symptoms
(Slide 27)
What do seizure symptoms depend on?
The part of the brain involved in the epileptic discharge
(Slide 27)
How long do most seizures last for?
~ 1 minute
(Slide 27)
What is epilepsy?
A syndrome of 2 or more unprovoked or recurrent seizures on more than 1 occasion
(Slide 28)
What are epileptic syndromes characterised by?
The behaviour during the seizures
The age of onset
Etiology (cause) if known
EEG characteristics of the seizure types
(Slide 28)
What is epilepsy which is at least partially resistant to drug treatment called?
Intractable epilepsy
(Slide 29)
What is the difference between focal and generalised epilepsy?
Focal epilepsy seizures begin from a single location within one cortical hemisphere of the brain whereas generalised epilepsy seizures originate from a deeper structure of the brain and project to both hemispheres simultaneously
(Slide 31)
What percentage of epilepsy does focal epilepsy make up?
80%
(Slide 32)
What 2 seizures are part of focal epilepsy?
Simple partial seizures (consciousness preserved)
Complex partial seizures (impaired level of consciousness)
(Slide 32)
What are 3 examples of generalised seizures?
Answers Include:
Absences
Myoclonic
Clonic
Tonic
Atonic
(Slide 32)
How does pharmacotherapy help treat epilepsy?
Reduces burst firing (by blocking sodium channels)
Increases inhibition (through GABA potentiation)
Reduces coupling (through a glutamate receptor antagonist)
(Slide 39)
