Module 7.3 Cancer Genome Sequencing 2 Flashcards
Liquid Biopsy
1
Q
Tissue Biopsy
drawbacks
A
- Invasive
- Longer time and higher cost for sample acquisition
- Information limited to sampling area and time
- Tissue sample not always available
- Not compatible with disease monitoring during surveillance/treatment
2
Q
Liquid Biopsy
features
A
- analyzes various bio markers present in body fluids (blood, urine, or CSF)
3
Q
Liquid Biopsy
benefits
A
- Minimally invasive
- Shorter time and lower cost for sample acquisition
- Comprehensive molecular information
- Enable testing in the absence of tissue
- Allow repeated sampling
- Enable disease monitoring during surveillance/treatment
4
Q
Liquid Biopsy
Applications (3)
A
Therapy selection
- can identify specific genetic mutations, copy number alterations, and other molecular changes associated with cancer
Minimal residual disease(MRD) detection and response monitoring
- can detect residual tumor cells (tumor DNA) that remain in bloodstream after surgery or other treatment
- monitor via serial liquid biopsies
- allows researchers to study clonal evolution during cancer progress
Early screen
5
Q
liquid biopsy biomarkers
cancer / non-cancerous tissues
A
From cancer:
* Protein
* Exosome
* Circulating tumor cells (CTC)
* Circulating tumor RNA
* Circulating tumor DNA (ctDNA) etc.
From non-cancerous tissue:
* Protein
* Exosome
* White blood cells (WBC)
* Red blood cells (RBC)
* Cell free RNA
* Cell free DNA (cfDNA) etc.
6
Q
Circulating tumor DNA
A
- Carries cancer specific variant information
- Short half-life (16 min - 2.5 hours) = real-time snapshot
- Extremely low quantity: nanogram (ng) level
- Short, fragmented DNA: ~170bp
- Low signal to noise ratio:
- rare tumor DNA in high non-tumor DNA
background (e.g. ≤0.1% mostly, 10% in late stage) - Sample prep, enzymatic and sequencing errors
( >0.1% NGS error rate) - need 0.1-0.5% VAF for NGS raw reads
7
Q
cell-free DNA
sources (3)
A
- apoptosis
- necrosis
- active secretion
8
Q
cell-free DNA
features
A
- detected in various body fluids (urine, saliva, etc.)
- carry genetic and epigenetic information from cell of origin.
- healthy: majority released from white blood cells
- great biomarker for therapy selection when tissue biopsy is not available
- most created through apoptosis and continue to be cleared from circulation via nuclease
9
Q
Unique Molecular Identifier
(UMI)
features
A
- string of oligonucletides that when attached to target DNA molecule, can be used as unique barcode to track particular DNA molecule throughout library preparation process and sequencing
- > 200 million possible UMI sequences
- reduce sequencing error frequency by 20 fold to about 10-4 to 10-5
- tags each strand of double stranded DNA independently
- sequencing must be deep enough to achieve multiple duplicated reads per UMI
- can remove errors introduced during late cycle PCR and sequencing.
- cannot remove errors or damages occurring before or during first two cycles of PCR
10
Q
Unique Molecular Identifier
(UMI)
process (PCR based)
A
- Forward primer: 5’ Universal Adapter 1, 14 degenerate bases (UMI), and 3’ gene-specific forward primer. Reverse primer: 5’ Universal adapter 2, 3’ gene-specific reverse primer
- 1st PCR cycle: forward UMI primers and reverse primer amplify original strands to create products with either universal adapter 1 w/ UMI or universal adapter 2
- 2nd PCR cycle: New forward UMI primers amplify UA2 strands and reverse primer amplify UA1 strands to create amplicons with both universal adapters and UMI.
- Sequence final PCR products (multiple duplicated reads)
- true mutation in original DNA template molecule will be present at every read with same UMI.
- Any variation in sequence of identically tagged reads = technical error
11
Q
Duplex sequencing
features
A
- true variant will have two complementary bases on both strands of one double stranded DNA
- DNA damage or artifacts likely to be present only in one strand
- tags both strands of duplex DNA with a random yet complementary double stranded nucleotide sequence (duplex tag)
- reduce error frequency to the range of 5x10-8
- need more than 6 reads per molecule for high accuracy
- 0.1% VAF = 3000 unique molecule x 10 reads = 30,000X coverage
12
Q
Duplex sequencing
process
A
- introduce a single stranded, random nucleotide sequence into one Y-adapter strand
- extend opposite strand with DNA polymerase to yield complementary double stranded tag (duplex tag: alpha or beta)
- ligate tagged adapters to T-tailed DNA fragment. Each end has either alpha or beta tag near Arm 1 (5’ end)
- Perform pair-end sequencing on flow cell to generate AB and BA strands for each fragment.
- identify and group members of each PCR family based on identical tag sequence.
- compare uniquely tagged PCR duplicates to create PCR consensus sequence. Only PCR families consisting of >3 duplicates and yielding same sequence in >90% of members at a given position used to create consensus sequence
13
Q
CHIP mutations
A
- Clonal Hematopoiesis of Indeterminate Potential
- presence of clonally expanded HSC cells, caused by mutations in individuals without evidence of hematologic malignancy
- HSCs gradually acquire somatic mutations as humans age and may gain competitive advantage eg. self-renew more often than HSCs without mutation
- confounding factor for accurate classification of somatic mutations.
- best to sequence tumor tissues or cell free DNA together with white blood cells so and only calls variants that are found in cell free DNA or tumor tissues, but not in white blood cell
14
Q
Hematopoietic Stem Cells
(HSC)
A
- multipotent cells that give rise to differentiated blood cells, including red blood cells, white blood cells, and platelets
- found in umbilical cord, blood, peripheral blood, and adult bone marrow
- able to self-renew aka replicate while still in undifferentiated state
15
Q
Minimal Residual Disease (MRD) Detection
A
- small amount of cancer cells that remain in person after treatment
- major cause of cancer relapse
- detection needed when tumor tissue already removed from patient after intended curative surgery or treatment
- blood sample collected when no visible tumor in patient
- tumor fraction in the plasma cell free DNA are often < 0.1%
