Exam 3 Flashcards
HPV, NGS, HLA, nanopore
true or false: all cells that express class 2 proteins also express class 1 proteins
true
What is the gold standard for HPV detection
molecular detection
ThinPrep PresercCyt cervical PAP smear
SurePath specimen
(both can used for cytology & testing)
Specimen types for HPV
alleles named in sequential order as they are discovered
EX: “A25:03” third specific allele, 03, of the HLA-A25 family of alleles
revised nomenclature for HLAs
how many molecules were sequences for given read length
read length
shows how many reads have accumulated per each of the detected barcode (indices) on the run
barcode hits
describes how much data has already been base called, how much has passed quality filters
cumulative output
How does read length impact sequencing analysis?
Longer reads improve genome assembly and structural variant detection, while shorter reads enhance accuracy in repetitive regions.
plates loaded w/ ab against known HLA types
the donor or recipient lymphocytes placed in wells
complement dependent cytotoxicity (CDC) test
how do different HLA typing methods compare in resolution?
Serological methods provide low resolution, PCR-based methods offer intermediate resolution, and sequencing methods provide high-resolution HLA typing.
-added to DNA fragments before PCR amp to remove duplicates
unique molecular identifiers
multiple sections of the circle representing the relative quantity of each organism
multiple source specimen
High cytotoxicity
cells have surface antigens matching the known antibody in the well
interpretation of CDC
-voltage is constant during sequencing but changes during pore scans
-voltage controls whether DNA molecules are pulled though pore
bias voltage history
incomplete reference genomics in the bioinformatics workflow
unknown calls
What is a Q-score in nanopore sequencing?
A Q-score indicates basecalling accuracy; a Q10 score equals 90% accuracy, while higher Q-scores imply greater confidence.
responsible for load of HPV in cervical cancer
E6 & E7
too many small fragments
too little DNA
poor adapter ligation
problem with the library
positive crossmatch
recipient serum kills donor lymphocytes
donor is not a possible candidate
interpretation of crossmatching
-visual representation of the taxonomic composition of the sample
-hierarchal ranking
-species on the outside layer
sunburst taxonomy graph
tissue and blood cells of a separate genetic origin
full chimera
non-enveloped, double-stranded circular DNA genome
-early expressed
-late expressed
-long control region
genome of HPV
Which transplant requires the highest level of HLA typing?
bone marrow
disrupts pRB, causing stimulation of cellular DNA synthesis and cell division
E7 protein
What are squiggles in nanopore sequencing?
Squiggles are electrical signal traces generated as DNA bases pass through the nanopore, used for basecalling.
Which two HLAs classes have antigen presenting proteins?
Class 1 and 2
What molecular methods are used to identify HLA types?
Techniques include sequence-specific oligonucleotide probe hybridization (SSOP), sequence-specific PCR (SSP-PCR), and direct DNA sequencing.
region of interest amplified using PCR
Low resolution
Sequence-specific oligonucleotide probe hybridization (SSOP)
Which transplant is associated with BK virus?
kidney
How are HLA alleles named?
The WHO nomenclature assigns names based on gene (e.g., HLA-A), followed by an asterisk and a numerical code indicating allele specificity (e.g., HLA-A*26:01).
the detection units in a flow cell, each containing multiple pores, but only one pore per channel is active at a time.
Channels
variant present in small proportion of the cell which indicates its not driving the growth of tumors
low VAF
If HPV is persistent what will it lead to?
cervival cancer
sequencing
available
recovering
inactive (dead pore)
channel classifications
more mapped reads= better identification confidence
number of reads per barcode
how fast the molecule is moving through the pore
translocation speed
all of the circle showing the same species
single source specimen
What’s include in FASTQC report include
-base sequence quality
-sequence quality scores
-base GC content
-base N content
-sequence length distribution
-sequence duplication levels
what can affect a Phred score
sequencing chemistry, read length, and the instrument used for sequencing
How are engraftment success and failure determined?
By analyzing the proportion of donor-derived DNA using STR analysis.
related to the accuracy of base calling
quality scores
recipient serum (antibodies) tested against donor lymphocytes
crossmatching
Cancers of the oropharynx, cervix, vulva, vagina, anus & penis
high risk HPV
how to calculate VAF
dividing the number of variant reads by the total number of reads
MUST BE EXPRESSED AS A PERCENT
1.Amplicons denatured and spotted onto a membrane
2.labeled oligonucleotide probes are designed to hybridize to specific HLA alleles
3. Spots detected from chemiluminescent or calorimetric signal
Steps for SSOP
50 pores
minimum QC criteria for a flongle
variant present in a large proportion of cells which indicates that its driving the growth of the tumor
high VAF
Highest resolution
distinguish between one-nucleotide difference
direct DNA sequencing
What can a Phred score exclude
low quality base calls that have high probability of being incorrect
What is the MHC locus and its function?
The MHC locus is a group of genes on chromosome 6 that encodes human leukocyte antigens (HLAs), which are essential for immune system function and transplant compatibility.
What does MinKNOW software monitor during sequencing?
It tracks sequencing quality, detects errors, and monitors trends like voltage, temperature, and pore status.
report shows quality information and gives read length and base yields
Metagenomics report
What do you want to do to PCR duplicates
remove them from the sequencing sample because it will lead to uneven distribution and artifacts will be removed
process of attaching functional information to the genetic sequences
variant annotation
What is a MUX/Pore scan?
A diagnostic scan that assesses active, recovering, and inactive pores in a sequencing run.
What type of resolution is needed for bone marrow/stem cell transplants
high resolution
set of particular alleles on the same chromosome
inherited together as a block
Haplotype
HLA inheritance
What are informative and non-informative loci?
Informative loci distinguish donor and recipient cells, while non-informative loci appear identical in both.
-viral capsid protein
-target for amp & detection for HPV
L1 gene
-amplifies only specific alleles
-control amplicons and allele-specific amplicons can be resolved by gel electrophoresis
sequence specific PCR (SSP-PCR)
HLA-A*26:03
high resolution
examines how many of the wells are one working pores, multiple working pores, zero pores, unavailable, or saturated
pore scan check
in the nucleus but outside the chromosome
benign HPV
allows for real-time base calling and alignment according to the desired bioinformatics pipeline
EPI2ME
reflects the balance between informative sequence data and the noise that is not informative or hard to interpret
signal to noise ratio
What is the role of alignment in sequencing?
Aligning reads to a reference genome helps identify mutations, structural variations, and microbial species.
What does high Phred score indicate
low probability of an incorrect base call
what are some problems with transplants
rejection and graft vs. host disease
High resolution
focus on most polymorphic loci in the MHC
use whole blood from EDTA tube
DNA-based typing
What strains are protected against in the HPV vaccine?
high and low risk strains
one read at a time, looking at reference genome & figuring out when read aligns
alignment
only recipient cells are found after transplant
failed transplant
Molecules expressed on the surface of all nucleated cells in the body + platelets
HLA class 1
What are ethical concerns in HLA typing and transplantation?
Issues include donor consent, false paternity discovery, and equitable organ allocation.
molecules expressed on the surface of “professional” antigen-presenting cells of the immune system (B-cells, lymphs, monocytes, macrophages, & dendritic cells)
HLA class 2
measure of how common a particular variant is within a population of cells
variant allele fraction (VAF)
What diseases are associated with MHC
autoimmune disease
measure of the quality of a base made by an NGS platform
Phred score
HLA-A*26
HLA-A
low resolution
How to determine who cells are whose?
STR analysis by fragment analysis
oncogene __ products interacts with p53
E6 gene
reagent serum for known anti-HLA antibodies are used that recognize specific HLA loci or antigens
traditional typing (serological analysis)
genital warts
low risk HPV
-used for sorting DNA/RNA sequences
-test based format for storing reads including quality values for each base
FASTQ
What is the role of the MDx laboratory in organ and bone marrow transplantation?
The MDx laboratory performs HLA typing to match donors and recipients, monitors engraftment success, and detects potential transplant rejection or graft-versus-host disease.
signal trace when each base interrupts the electrical potential of the membrane
squiggles
How is STR analysis used for post-transplant monitoring?
Short Tandem Repeat (STR) analysis differentiates donor and recipient DNA to assess engraftment success.
What are some reasons for non-uniform coverage?
sequencing bias, library preparation bias, mapping bias, sampling bias, experiment design
detect patients who are at risk for developing cervical dysplasia and cervical cancer
HPV clinical utility
donor and recipient cells are in found in patient sample
mixed chimera
individual with genetically distinct cell populations from donor and recipient sources
chimera
What is full chimerism vs. mixed chimerism?
Full chimerism means only donor cells are detected, while mixed chimerism indicates a mix of donor and recipient cells.
cells from terminal state go to active state
viral replication
-dont test below age 25
-cant distinguish between transient & persistent infections
-HPV does not = cervical cancer
-controversy: HPV is the sole screening method for cervical cancer risk
HPV testing considerations
voltage issues
air bubbles
temp fluctuations
problems with a flow cell
What is translocation speed?
The speed at which DNA molecules pass through a nanopore, affecting sequencing efficiency and accuracy.
basal cells to mature keratinocytes
-viral DNA is replicated
-capsid protein synthesized
-viral DNA release occurs
HPV life cycle
what will cause low signal to noise ratio
background noise is loud meaning artifacts are being sequenced
What factors can cause errors in HLA typing?
Contamination, low DNA quality, ambiguous allele calls, and reagent issues can affect results.
What are the genomic features of HPV?
HPV is a non-enveloped, double-stranded circular DNA virus with early-expressed genes, late-expressed genes, and a Long Control Region (LCR).
What are the most common high-risk HPV strains?
HPV 16 and HPV 18 are the most common high-risk strains associated with cervical cancer.
What is the role of the E6 and E7 genes in HPV infection?
E6 degrades the tumor suppressor p53, while E7 disrupts the retinoblastoma protein (pRB), leading to uncontrolled cell division.
How does HPV progress from infection to cancer?
HPV initially exists in an episomal state but integrates into the host genome in advanced stages, leading to cancerous transformation.
What are the molecular methodologies used for HPV testing?
Molecular methods include hybrid capture (signal amplification), transcription-mediated amplification, and real-time multiplex PCR for strain typing.
What are the clinical applications of HPV testing?
HPV testing is used for cervical cancer risk assessment, primary screening, and co-testing with Pap smears.
What are the strengths of HPV testing?
High sensitivity, ability to detect high-risk strains, and strong predictive value for cervical dysplasia.
What are the limitations of HPV testing?
Cannot distinguish between transient and persistent infection, and a positive result does not guarantee cancer.
What are the specimen types used for HPV testing?
ThinPrep and SurePath cervical Pap smear specimens.
Why is HPV testing not recommended for individuals under 25?
HPV infections are common and often clear naturally in younger individuals without causing cervical dysplasia.
component of a FASTQ report
read identifier
nucleotide sequence
confidence score
total number of reference sequence bases for
which reads from a sample have been aligned
coverage
