Midterm 2 - Notes 5 (Part 2) Flashcards
What 3 signals help to regulate the cell cycle?
- GPCR
- Protein phosphatase
- Nucleotide exchange factor
What 2 ways can transport proteins?
- Peptide/drug transporter
2. Cadherin family protein
What does the 2nd case study describe?
Whole genome cancer approach
- mutation evolution in a lobular breast tumour profiled at single nucleotide resolution
What type of tools did they use on metastatic tumours? (3)
- Whole genome sequencing
- Transcriptome sequencing
- Illumia sequencing
What was the control sample in the 2nd case study?
Blood sample from same patient
- provides reference genome
Why were the lengths made longer?
To make it easier to detect differences
What could the tumour transcriptome not do?
Give coverage
What were the mutations likely caused by?
Somatic tumours
How can you tell if its a somatic mutation or not?
See if it was present in the reference genome
What indicates the expression level of a gene?
The number of genes that are counted
How can you tell if there is over/under expression in a gene?
Compare it to the reference genome
What can changes of expression be caused from?
Epigenetic change
What did they use in the experimental design?
Used whole genome sequencing to look for differences and re-arrangements
- didnt use whole genome sequencing on the control
What did they use for the control instead of whole genome sequencing?
Used a pipeline to identify candidate differences in protein coding genes and then went back to the target to use PCR and Sanger sequencing to see what the differences were
What did they develop to detect alterations?
Algorithms
What were the alterations tested? (4)
- Insertions/deletions
- Inversions
- Gene fusions
- Copy number alterations
- used FISH for this one
What was the easiest thing to detect?
SNVs
What did they remove from the SNVs? (2)
- Pseudogenes
2. HLA sequences
When they re-sequenced, what did they use?
Sanger
What were they surprised they found in the 2nd case study?
Finding 5 transcriptomes in the sequencing data
Were somatic mutations identified?
Yes
What did they want to identify?
If the mutation actually was present in a few cells in the primary tumour and if so how many in the cell had these mutations
- 5 of them (most common)
What happens if you amplify these genes?
99% of them dont have the mutations, the PCR products will have the WT base and only 1% will have the mutated base at this position
What did they use for sequencing? And what did they use to confirm the sequencing?
Used Illiuma to sequence and Sanger to confirm
What is an example of an RNA enzyme?
ADAR
Where is ADAR expressed?
It is highly expressed in tumour samples
What does ADAR mediate?
A –> G edits in target RNA
What is highly regulated?
RNA editing
What did they discover?
That performing NGS on tumours is feasible
