Genetic Testing Flashcards
How many bases in the human genome?
~3 billion
How many protein coding genes are in the human genome?
20k (1.5% of the genome)
Clinical utility of a genetic test
Evidence of an improved measurable clinical outcome
Clinical validity of a genetic test
Relationship between a test result and disease
Analytical validity
Test accuracy
Genetic variation
Variations in:
- sequence
- copy number
- expression
- epigenetic modifications
Targeted vs nonspecific fluorescent detection methods for RT-PCR
- SYBR green dye binds any DNA
- Probes (Taqman, molecular beacons, light cycler) require a specific sequence to fluoresce
PCR pros/cons
- Pro: low limit of detection
- Con: requires specific primer or probe design => only used to detect known variants
Methods for detecting novel sequence variation
- Sanger sequencing
- Pyrosequencing
- NGS
Sanger dideoxy sequencing
- 10% of nucleotides will be fluorescently labelled ddNTPs
- As polymerase extends a sequence, chain terminating ddNTPs will be incorporated
- Fragments will be ordered by length via gel electrophoresis
- Colors read off in order => sequence
Limitations of sanger sequencing
- cis-trans ambiguity
- not quantitative
- 10-20% allele frequency limit of detection
- primers designed to include only coding sequences and splice sites
- large detections on only 1 allele won’t be detected
Cis-trans ambiguity
Inability to determine which allele a variation corresponds to: if there is a sequence variation at position 1 and position 13, wouldn’t know if they are on the same chromosome or different copies
Pyrosequencing
- dNTPs washed over flow cell one at a time
- if a dNTP is incorporated into a growing strand by polymerase, PPi is released
- PPi is used to generate ATP by sulfurylase
- ATP is converted to light by luciferase
- light = that nucleotide was incorporated
Pros/cons of pyrosequencing
Pro = quantitative (double light = two bases) Con = short read length
NGS
Pyrosequencing in parallel!
- Create library by enriching for target DNA and ligating on adaptors
- Capture fragments on a flow cell and amplify to generate clusters of identical strands
- Wash dNTPs over the flowcell, imaging after each addition to see which bases were added to which clusters
- Trim, align, analyze
Pros/cons of NGS
Pros = quantitative, low limit of detection, remove cis-trans ambiguity, decreased cost Cons = require sufficient depth to differentiate errors from variation, some regions will not be covered (deep vs broad coverage), certain mutations not detectable (highly repetitive/homologous regions)
SNP frequency
variant with frequency > 1% in the population
Types of variants
- substitutions (transitions vs transversions)
- deletions
- insertions
- inversions
Missense substitution
Codes for a different AA
Nonsense substitution
Gain of stop
Synonymous/silent substitution
- Codes for same AA
- May affect a splice site
Splice effects from substitutions
- Intronic splice silencer
- Intronic splice enhancer
- Exonic splice silencer
- Exonic splice enhancer
In-frame indels
Addition of a multiple of 3 nts maintains reading frame
Frameshift indels
Addition of not multiples of 3 shift reading frame
DNA-level variant nomenclature
c.76A>T
RNA-level variant nomenclature
r.76a>u
Protein-level variant nomenclature
p.Lys76Asn
Coding DNA
exons and splice sites only
Genomic DNA
sequence as present in the genome
