Genetic Testing

Question 1

How many bases in the human genome?

Answer 1

~3 billion

Question 2

How many protein coding genes are in the human genome?

Answer 2

20k (1.5% of the genome)

Question 3

Clinical utility of a genetic test

Answer 3

Evidence of an improved measurable clinical outcome

Question 4

Clinical validity of a genetic test

Answer 4

Relationship between a test result and disease

Question 5

Analytical validity

Answer 5

Test accuracy

Question 6

Genetic variation

Answer 6

Variations in:

• sequence
• copy number
• expression
• epigenetic modifications

Question 7

Targeted vs nonspecific fluorescent detection methods for RT-PCR

Answer 7

• SYBR green dye binds any DNA

- Probes (Taqman, molecular beacons, light cycler) require a specific sequence to fluoresce

Question 8

PCR pros/cons

Answer 8

• Pro: low limit of detection

- Con: requires specific primer or probe design => only used to detect known variants

Question 9

Methods for detecting novel sequence variation

Answer 9

1. Sanger sequencing
2. Pyrosequencing
3. NGS

Question 10

Sanger dideoxy sequencing

Answer 10

• 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

Question 11

Limitations of sanger sequencing

Answer 11

• 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

Question 12

Cis-trans ambiguity

Answer 12

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

Question 13

Pyrosequencing

Answer 13

1. dNTPs washed over flow cell one at a time
2. if a dNTP is incorporated into a growing strand by polymerase, PPi is released
3. PPi is used to generate ATP by sulfurylase
4. ATP is converted to light by luciferase
5. light = that nucleotide was incorporated

Question 14

Pros/cons of pyrosequencing

Answer 14

Pro = quantitative (double light = two bases)
Con = short read length

Question 15

NGS

Answer 15

Pyrosequencing in parallel!

1. Create library by enriching for target DNA and ligating on adaptors
2. Capture fragments on a flow cell and amplify to generate clusters of identical strands
3. Wash dNTPs over the flowcell, imaging after each addition to see which bases were added to which clusters
4. Trim, align, analyze

Question 16

Pros/cons of NGS

Answer 16

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)

Question 17

SNP frequency

Answer 17

variant with frequency > 1% in the population

Question 18

Types of variants

Answer 18

• substitutions (transitions vs transversions)
• deletions
• insertions
• inversions

Question 19

Missense substitution

Answer 19

Codes for a different AA

Question 20

Nonsense substitution

Answer 20

Gain of stop

Question 21

Synonymous/silent substitution

Answer 21

• Codes for same AA

- May affect a splice site

Question 22

Splice effects from substitutions

Answer 22

• Intronic splice silencer
• Intronic splice enhancer
• Exonic splice silencer
• Exonic splice enhancer

Question 23

In-frame indels

Answer 23

Addition of a multiple of 3 nts maintains reading frame

Question 24

Frameshift indels

Answer 24

Addition of not multiples of 3 shift reading frame

Question 25

DNA-level variant nomenclature

Answer 25

c.76A>T

Question 26

RNA-level variant nomenclature

Answer 26

r.76a>u

Question 27

Protein-level variant nomenclature

Answer 27

p.Lys76Asn

Question 28

Coding DNA

Answer 28

exons and splice sites only

Question 29

Genomic DNA

Answer 29

sequence as present in the genome