Genet 302 lec 14 & 15
What was the main limitation of Sanger sequencing compared to next-generation sequencing?
Sanger sequencing is best for small targets (single genes, PCR products, plasmids), whereas next-generation sequencing can handle whole genomes and communities.
Around what year did next-generation sequencing become widely used?
Around 2008.
What types of samples can Illumina MiSeq sequence?
Individual genomes (genomics) and environmental samples (metagenomics).
What is the approximate cost of running an Illumina MiSeq experiment?
$1000+.
What is the first step in the Illumina MiSeq workflow?
Break the DNA sample into random fragments.
What device can be used to shear DNA into fragments?
A Covaris g-TUBE.
What is Step 2 in the Illumina MiSeq workflow?
Verify fragment lengths.
What instrument is used to verify DNA fragment size?
Agilent 2100 Bioanalyzer.
What was the average fragment length shown in the lecture example?
408 bp.
What is Step 3 in the Illumina MiSeq workflow?
Load the DNA library into a cartridge.
Where does DNA sequencing occur in the MiSeq system?
On a sequencing chip inside the instrument.
What format is MiSeq output initially provided in?
Raw sequence data (large blocks of A, C, G, T).
What is personalized genomics?
Using genome sequencing to understand disease risk or future health conditions in an individual.
What is the purpose of parent-of-origin detection?
To determine whether a mutation is on the maternal or paternal chromosome.
What is a haplotype?
The set of alleles present on one chromosome inherited from one parent.
Why can next-gen sequencing help resolve haplotypes?
It can sequence long fragments that link variants together on the same chromosome.
Which chromosomes typically have more DNA methylation?
Maternal chromosomes.
What is 5-base DNA sequencing?
Sequencing that reads A, C, G, T, and methylated cytosine (Me-C).
What is the typical two-step workflow for genetic and epigenetic sequencing?
Step 1: sequence non-modified DNA for genetic information; Step 2: modify DNA and sequence for methylation information.
In bisulfite sequencing, what happens to unmethylated cytosines?
They are converted to thymine.
In methylation sequencing, what happens to methylated cytosines?
They remain distinguishable from unmethylated cytosines.
How can genetic sequencing and methylation sequencing be combined?
To assign mutations to maternal or paternal chromosomes.
Which syndrome example was used for maternal deletion detection?
Angelman syndrome del(15)(q11q11)mat.
What is Technology #2 discussed in this lecture?
MinION flow cell.
What is the first step when using a MinION device?
Prepare DNA in a PCR machine.
What is the second step when using a MinION device?
Inject DNA into the device.
What is the primary use of MinION sequencing?
Small genomes such as bacteria and viruses.
What is a major advantage of the MinION device?
It is very small and portable.
What is Technology #3 discussed in this lecture?
Plasmidsaurus.
What types of DNA can Plasmidsaurus sequence?
Plasmids, PCR products, bacterial chromosomes, and other small DNA molecules.
What is Step 1 of the Plasmidsaurus workflow?
Obtain the DNA to sequence (plasmid or PCR product).
What is Step 2 of the Plasmidsaurus workflow?
Mail the DNA sample to Plasmidsaurus.
How often does MBSU mail Plasmidsaurus samples?
Once a week.
What is the approximate machine cost for Plasmidsaurus sequencing?
$1000.
What is the approximate price per sample for users?
$15.
Why is Plasmidsaurus sequencing inexpensive?
Sample indexing allows many DNA samples to be combined and sequenced together.
What is sample indexing?
Adding unique identifier sequences to samples so they can be pooled and later distinguished.
In what format does Plasmidsaurus return sequencing results?
Chromatograms.
What must you know for the exam from this lecture?
Which sequencing technology is best for a specific experiment, the parent-of-origin procedure, and the Plasmidsaurus workflow.
What is the main purpose of a DNA microarray?
To determine copy number changes and identify which SNP alleles are present at specific locations.
What does SNP stand for?
Single nucleotide polymorphism.
In the context of microarrays, what does “probe” refer to?
A short oligonucleotide sequence attached to a glass or silicon surface that hybridizes to target DNA.
What is a DNA microarray?
A collection of oligonucleotide probes attached to a solid surface used to detect complementary fluorescently labeled target DNA.
What is Step 1 of the DNA microarray procedure?
Fluorescently label the target DNA.
What is Step 2 of the DNA microarray procedure?
Add the labeled DNA to the microarray.
What determines which microarray spots light up?
Complementary hybridization between target DNA and probe sequences.
What is a tiling microarray?
A microarray where spots represent regions across chromosomes.
What are tiling microarrays used for?
Applications such as aCGH and ChIP.
What is a genotyping microarray?
A microarray where spots represent SNPs.
What are genotyping microarrays used for?
GWAS and consumer testing like 23andMe.
What is the difference between one-channel and two-channel microarrays?
One-channel uses one fluorescent dye per sample; two-channel compares two samples using different dyes (e.g., red and green).
Which applications commonly use one-channel microarrays?
GWAS.
Which applications commonly use two-channel microarrays?
aCGH.
What are pre-made DNA microarrays?
Commercially produced arrays designed for common applications such as human genotyping.
What is an example of a pre-made genotyping array?
Illumina Infinium Global Screening Array.
How many SNP markers can large commercial genotyping arrays analyze?
Hundreds of thousands to millions of SNPs.
What are custom-made DNA microarrays?
Arrays designed for specific research purposes with selected SNPs or regions.
What facility at the University of Alberta helps with microarray work?
MBSU (Molecular Biology Service Unit) in CCIS.
In the polar bear example, what was Step 1?
Sequence various polar bears.
What was Step 2 in the polar bear example?
Use software to identify 9000 SNPs.
What was Step 3 in the polar bear example?
Work with Illumina to design a custom two-channel SNP microarray.
What was the approximate cost per custom microarray in the example?
$1440.00.
What device is used to scan microarrays after hybridization?
A microarray scanner.
What software is used to analyze SNP and CNV data from microarrays?
GenomeStudio software.
What type of data visualization was shown for polar bear population structure?
Principal component analysis (PCA) plot.
What categories of DNA microarrays must you know for the exam?
Pre-made, custom-made, or locally-made; tiling or genotyping; one-channel or two-channel.