Lecture #8 Flashcards
PGx testing methods
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
Polymerase chain reaction
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)
PCR method
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
PCR
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
Important point
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
DNA chip
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
DNA (sanger) sequencing
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)
Sanger sequencing is a method of DNA sequencing based on the
selective incorportation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerasae during in vitro DNA replication
How to read sanger sequencing
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
Sanger sequencing can detect
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
Next generation sequencing
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
Sequencing depth and coverage
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
For detecting human genome mutations, SNPs, and rearrangements…
10x-30x depth of coverage is often recommended, depending on the application and statistical model
next generation shotgun sequencing approached require sequencing every base in a sample several times for two reasons:
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
4x sequencing depth
cannot determine is heterozygous or homozygous
10x sequencing depth scenario - 5 of “C” read
more than likely the pt is heterozygous for this SNP
10x sequencing depth - 9 of “C” read
more than likely this pt is homozygous for this allele
Germline vs somatic
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
Detection methods for somatic mutations
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
HIPPA
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
