Lecture #8

Question 1

PGx testing methods

Answer 1

goal of the testing: testing the known variants - genotyping: DNA chip; testing both known and unknown alleles - sequencing: sanger sequencing, high-throughput next-gen sequencing (whole exome sequencing)
fundamental technique for DNA amplification: PCR

Question 2

Polymerase chain reaction

Answer 2

most useful technique for DNA amplification: 50-1000 bp
amplify a specific region from the genome for making billions of copies: detectable
enzymatic reaction
substrates: DNA template, dNTPs (dATP, dGTP, dCTP, dTTP), primers: 2 short sequences specific to the region of interest, buffer: pH, Mg2+, enzymeL Taq DNA polymerase
products: DNA molecules (fragments start and end with primers)

Question 3

PCR method

Answer 3

denaturation: temperature is increased to separate DNA strands
annealing: temperature is decreased to allow primers to base pair to complementary DNA template
extension: polymerase extends primer to form nascent DNA strand
exponential amplification: process is repeated and region of interest is amplified exponentially

Question 4

PCR

Answer 4

a chain reaction from 2 copies to 2^n+1 copies (n = # of thermal cycles)
starts from very small amount of template DNA
enzyme (Taq polymerase) is the key: thermal stable, isolated from thermus aquaticus

Question 5

Important point

Answer 5

PCR amplifies DNA from both DNA molecules of homologous chromosomes
this is why you can tell a genotype
PCR reaction products (amplicon) are a mix of double-strand DNA products generated from both homologous chromosomes (primers equally bind to each chromosome): we need additional specific technique to distinguish each allele

Question 6

DNA chip

Answer 6

detecting known SNPs or targeted SNPs
high throughput: up to 5M SNPs can be genotyped simultaneously
medium cost: low per SNP cost
large-scale used for research: genome-wide based studies
mid-throughput use for PGx testing

Question 7

DNA (sanger) sequencing

Answer 7

several methods have been developed for DNA sequencing: conventional sequencing (sander sequencing) - low throughput, targeted sequencing; next generation sequencing - high throughput sequencing, parallel sequencing, massive seqeuncing (sequencing multiple DNA fragment simultaneously)

Question 8

Sanger sequencing is a method of DNA sequencing based on the

Answer 8

selective incorportation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerasae during in vitro DNA replication

Question 9

How to read sanger sequencing

Answer 9

A tube, G tube, C tube, T tube, read from bottom right to the left going up
chain termination at different locations because the insertion is random, longer the sequence, heavier the molecule is

Question 10

Sanger sequencing can detect

Answer 10

both known and unknown alleles: SNPs, indel, small CNV
low throughput: 96 samples per overnight for one DNA fragment ~700 bp
relatively higher cost per base pair, high per SNP cost
widely used in PGx testing

Question 11

Next generation sequencing

Answer 11

sequencing by the synthesis in parallel
high throughput, can simultaneously sequence DNA of multiple individuals
customizable capacities: whole genome/whole exome (all exon sequences) vs targeted genes
higher total cost
very low cost per SNP
detect all known or unknown alleles
detect almost all kinds of polymorphisms
increasing use in clinical PGx testing: diagnosis to customer, peronsalized PGx discovery

Question 12

Sequencing depth and coverage

Answer 12

the average # of reads that align to, or cover known reference bases
next generation sequencing coverage level often determines whether variant discovery can be made with a certain degree of confidence at particular base positions
specific depth to tell what’s just a mistake in the sequencing vs what’s an actual mistake

Question 13

For detecting human genome mutations, SNPs, and rearrangements…

Answer 13

10x-30x depth of coverage is often recommended, depending on the application and statistical model

Question 14

next generation shotgun sequencing approached require sequencing every base in a sample several times for two reasons:

Answer 14

you need multiple observations per base to come to a reliable base call
reads are not distributed evenlt over an entire genome, simply because the reads will sample the genome in a random and independent manner

Question 15

4x sequencing depth

Answer 15

cannot determine is heterozygous or homozygous

Question 16

10x sequencing depth scenario - 5 of “C” read

Answer 16

more than likely the pt is heterozygous for this SNP

Question 17

10x sequencing depth - 9 of “C” read

Answer 17

more than likely this pt is homozygous for this allele

Question 18

Germline vs somatic

Answer 18

germline: sequence of germ cells that may be passed to a child: exists in the somatic genome, exists since the individual was born
somatic: sequence of nongermline cells that is not passed to a child: does not exist in the germline genome, acquired

Question 19

Detection methods for somatic mutations

Answer 19

sanger sequencing: point mutations, small indels
DNA chips usually not used for somatic mutation detection
NGS: almost all kinds of mutations
other methods: karyotyping, immunohitobiochemistry

Question 20

HIPPA

Answer 20

health insurance portability and accountability act: same as other medical info - lifelong issue: diff from other medical info; data sharing: genetic info non discrimination act (GINA), no discrimination, with other family members?
report: explanation for the genotype-phenotype association, potential risks and limitations