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?

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1
Q

How are normal cells different than cancer cells?

A
  • 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
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2
Q

What are the three stages of cancer?

A
  • Elimination by the immune system
  • Equilibrium is kept by the immune system, tumor is dormant
  • Escape from the immune system, progressive disease
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3
Q

How did the original sequencing occur?

A

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
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4
Q

What happens if there is no 3’-OH?

A
  • 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
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5
Q

Why is Sanger sequencing helpful?

A
  • 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
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6
Q

How was Sanger sequencing updated?

A
  • 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
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7
Q

How has sequencing the human genome changed in the past 10 years?

A
  • 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
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8
Q

How is the genomic DNA library prepared for Next Generation Sequencing?

A
  • 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
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9
Q

What does a flow cell look like?

A
  • There are 8 channels

- The surface of the flow cell is coated with a lawn of oligo pairs

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10
Q

How does cluster generation begin?

A
  • 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
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11
Q

After the formation of the newly synthesized strand, what occurs during Next Generation?

A
  • The double-stranded molecule is denatured
  • The original template is washed away
  • The newly synthesized strand is covalently bound to the flow cell surface
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12
Q

What is bridge amplification in Next Gen sequencing?

A
  • 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
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13
Q

What occurs in Next Gen once bridge amplification is completed?

A
  • 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
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14
Q

How does the sequencing cycle in Next Gen occur?

A
  • Determine the first base by adding the four labelled dNTPs

- Excited by a laser and the bases light up in their location

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15
Q

How are gaps filled in Next Gen?

A

Using a reference sequence, the fragment sequences are aligned and the gaps are filled by overlays

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16
Q

What is high coverage?

A

When there is a large amount of the genome sequenced

  • multiple fragments that cover various parts of the genome
  • more accurate
17
Q

What is low coverage?

A

When there is a small amount of genome sequenced

- could leave gaps

18
Q

What are the differences between Sanger Sequencing and Next generation Sequencing?

A
  • 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
19
Q

What could be missed from Whole-exome sequencing?

A

You would miss regulatory elements, such as microRNA, start sites, etc, that are within introns

20
Q

What is whole-exome sequencing used for?

A
  • point mutations

- copy number variations

21
Q

What is exome sequencing?

A
  • sequences only protein-encoding genes (exons, 1% of the genome)
  • looking at exons because they’re responsible for phenotypes in a cell
22
Q

What are the limitations of exome sequencing?

A
  • only accounts for 1% of the genome (~180K exons)

- misses deletions, variants and gene-rearrangements

23
Q

What are the advantages to exome sequencing?

A
  • cost effective
  • quick
  • can see point mutations
  • can look at copy number variations
24
Q

What is upregulated by at T-cell that has become active?

A
  • 4-1BB

- IFNg

25
Q

Why is personalized medicine important?

A
  • all cancers are genetically different with different mutations
26
Q

How is a person with metastatic cholangiocarcinoma treated with T cells? What are the results?

A
  • 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
27
Q

What cancer applications are there that have T cells that identify targets?

A
  • Adoptive T cell therapy
  • Vaccines
  • Chimeric antigen receptors
  • Engineered T cells