Lecture 25 Flashcards
- understand basic sequencing technology - what is exome sequencing and how is it different from other types of sequencing? - what information does exome sequencing provide? - how can exome sequencing data be applied and used for T cell therapy in cancer?
How are normal cells different than cancer cells?
- Cancer cells have a large, variably shaped nuclei
- Cancer cells divide frequently in a disorganized arrangement
- Cancer cells vary in size and shape
- Cancer cells lose normal features
What are the three stages of cancer?
- Elimination by the immune system
- Equilibrium is kept by the immune system, tumor is dormant
- Escape from the immune system, progressive disease
How did the original sequencing occur?
Sanger sequencing
- A single stranded DNA is labelled with a primer.
- DNA polymerase adds the complementary base from the excess dNTPs
- Each binding of dNTP stops the reaction -various sized fragment
- the fragments are run on a gel based on sizes
- gel is red from 5’ to 3’ end to determine the sequence
What happens if there is no 3’-OH?
- 3’-OH required for chain elongation
- if there is no 3’-OH then the chain terminates
- bases are added to the 3’ end
- could be removed due to mutation
Why is Sanger sequencing helpful?
- Allows you to see mutations which are visualized on the gel
ie. a heterozygous point mutation where there is a wild-type copy of the gene and a mutant copy of the gene in the same location
How was Sanger sequencing updated?
- The dNTPs were labelled with fluorescence
- The fluorescence is then detected by a computer
- Peaks are generated which correspond to a base
- Can sequence 1000 bases with accuracy
How has sequencing the human genome changed in the past 10 years?
- In 2006, the cost to sequence a single human genome was $10 million
- In 2015, to sequence more than 10 million human genomes cost less than $100
How is the genomic DNA library prepared for Next Generation Sequencing?
- The DNA fragment has its ends sheared, then polished to make them blunt ended
- The fragment is ligated to adapters
- It is then separated on a gel then cut out
What does a flow cell look like?
- There are 8 channels
- The surface of the flow cell is coated with a lawn of oligo pairs
How does cluster generation begin?
- Begins with hyridization of the fragment to the oligos
- > 100M single molecules hybridize to the lawn of primers
- Bound molecules are then extended by polymerases
After the formation of the newly synthesized strand, what occurs during Next Generation?
- The double-stranded molecule is denatured
- The original template is washed away
- The newly synthesized strand is covalently bound to the flow cell surface
What is bridge amplification in Next Gen sequencing?
- Single strand flips over to hybridize to adjacent primers to form a bridge
- Hybridized primer is extended by polymerases
- Double-stranded bridge is formed
- “PCR on a solid support” -> generates thousands of copies to provide enough signal during sequencing
What occurs in Next Gen once bridge amplification is completed?
- The double stranded bridge is denatured
- Results in two copies of covalently bound single-stranded templates
- these templates undergo the bridge amplification again until multiple bridges are formed
- they are then denatured and the reverse strands cleaved and washed away
How does the sequencing cycle in Next Gen occur?
- Determine the first base by adding the four labelled dNTPs
- Excited by a laser and the bases light up in their location
How are gaps filled in Next Gen?
Using a reference sequence, the fragment sequences are aligned and the gaps are filled by overlays
What is high coverage?
When there is a large amount of the genome sequenced
- multiple fragments that cover various parts of the genome
- more accurate
What is low coverage?
When there is a small amount of genome sequenced
- could leave gaps
What are the differences between Sanger Sequencing and Next generation Sequencing?
- Sanger uses a primer while NG uses oligos
- Sanger individually interogates each terminated DNA while NG uses a DNA library
- Less info is gathered by Sanger and NG gathers more info
What could be missed from Whole-exome sequencing?
You would miss regulatory elements, such as microRNA, start sites, etc, that are within introns
What is whole-exome sequencing used for?
- point mutations
- copy number variations
What is exome sequencing?
- sequences only protein-encoding genes (exons, 1% of the genome)
- looking at exons because they’re responsible for phenotypes in a cell
What are the limitations of exome sequencing?
- only accounts for 1% of the genome (~180K exons)
- misses deletions, variants and gene-rearrangements
What are the advantages to exome sequencing?
- cost effective
- quick
- can see point mutations
- can look at copy number variations
What is upregulated by at T-cell that has become active?
- 4-1BB
- IFNg
Why is personalized medicine important?
- all cancers are genetically different with different mutations
How is a person with metastatic cholangiocarcinoma treated with T cells? What are the results?
- A biopsy was taken of the patient’s cancer
- extracted T cells that recognized the tumor and grew up the numbers
- The mutation was found in the ERBB2IP protein by T cells
- Results: T cell therapy induced regression at 6 months but post 6 months there was an increase in tumor burden again. This could be due to other mutations
What cancer applications are there that have T cells that identify targets?
- Adoptive T cell therapy
- Vaccines
- Chimeric antigen receptors
- Engineered T cells
