BIOSC 1540: L02A (Sequencing)

Computational Biology

(BIOSC 1540)

Jan 14, 2025

Lecture 02A

DNA sequencing

Foundations

Announcements

Assignment P01A is due Friday (Jan 17) by 11:59 pm
Quiz 01 is in two weeks (Jan 28) and will cover from Lecture 02A to 03B

After today, you should have a better understanding of

Importance and applications of DNA sequencing

DNA sequencing revolutionizes biology and medicine through diverse applications

Medicine: Enables precision medicine, genetic disease diagnosis, and cancer genomics.
Agriculture: Enhances crop improvement, pest resistance, and livestock genetics.
Evolution: Deciphers evolutionary relationships and molecular phylogenies.
Microbiology: Identifies pathogens and studies microbial communities (e.g., metagenomics).
Ecology: Monitors biodiversity and tracks species in ecosystems.

After today, you should have a better understanding of

Techniques for extracting and purifying high-quality DNA

DNA extraction

How do we acquire our DNA sample?

Computationalists need to understand the underlying source of our data for quality control

Let's start with a bacterial culture

Fun fact: Pitt has a beer brewing class (ENGR 1933)

We let our bacterial culture produce our products of interest

Biotechnology frequently uses massive E. coli cultures to produce bioproducts

Separate cells from media

Great! We have our cells, but how can we get DNA out of our cells?

The first step is always to centrifuge and separate our cells and media

Keep the part that has our component of interest (DNA)

We break open our cells by lysing them

Chemical lysis destabilizes the lipid bilayer and denatures proteins

Surfactants have a hydrophilic head and hydrophobic tail

Wait, surfactants sound a lot like phospholipids?

What's the primary difference, and how does this change its behavior?

Surfactants possess a single hydrophobic tail. Why does the incorporation of these surfactants destabilize the phospholipid membrane?

Please note: TopHat questions are ungraded. Engaging honestly with the question will benefit you far more than any shortcuts.

After today, you should have a better understanding of

Techniques for extracting and purifying high-quality DNA

DNA purification

At this stage, we need to separate DNA from other biomolecules ... how?

We need to exploit physicochemical property differences (such as solubility, charge, and hydrophobicity) to separate DNA from other biomolecules

Phenol-chloroform extraction exploits solubility and density differences

Phosphate backbone
(negative charged)

Denatures and aggregates at interface

Phenol

Chloroform

Water

Nonpolar

DNA

RNA

Protein

Lipids

Collecting our aqueous phase selects only DNA and RNA

Silica column-based purification relies on ionic interactions

Under high-salt conditions, negatively charged DNA binds to the positively charged silica membrane via electrostatic interactions

Contaminants like proteins and salts do not bind or are washed away

DNA is then eluted with a low-salt buffer or water

Magnetic beads rely on selective adsorption and surface chemistry

Magnetic beads coated with DNA-binding agents (e.g., silica or polymer) selectively adsorb DNA in the presence of binding buffers

Magnetic fields are used to separate beads with bound DNA from the solution, allowing for washing away impurities like proteins, RNA, and salts

Note: Nowadays, most labs use highly effective kits

After today, you should have a better understanding of

Techniques for extracting and purifying high-quality DNA

DNA quality quantification

Before sequencing our sample, we should check the quality

DNA

Likely contaminants

RNA contamination can inflate DNA quantification readings due to similar properties

RNA

Protein

Why it's a problem

Proteins can inhibit enzymatic reactions in library preparation and distort DNA quantification

UV radiation is selectively absorbed based on molecular structure

Molecules with aromatic rings absorb UV light strongly due to their conjugated π-electron systems

UV light excites electrons in the π-bonds of aromatic systems to higher energy states

UV radiation is selectively absorbed based on molecular structure

Proteins absorb UV light primarily at 280 nm, mainly due to aromatic amino acids

DNA and RNA absorb UV light at 260 nm because their bases contain highly conjugated double bonds

A260/A280 ratio relates to sample purity

After today, you should have a better understanding of

Steps in preparing DNA libraries for sequencing

A DNA library is a collection of DNA fragments ready for sequencing

Fragmentation breaks DNA into smaller, manageable pieces

Methods include

Mechanical shearing (e.g., sonication)
Enzymatic digestion using restriction enzymes

Long DNA molecules cannot be sequenced by most platforms due to size constraints

DNA is fragmented to an optimal size range (e.g., 200–500 bp) for efficient sequencing and alignment

Adapter ligation enables amplification and sequencing

Adapters are short, synthetic DNA sequences that are ligated to the ends of DNA fragments during library preparation

PCR amplification ensures sufficient DNA for sequencing

During next-generation sequencing library preparation, short “adapter” sequences are added to the ends of DNA fragments. Which of the following best describes the primary reason for adding these adapters?

A. To link multiple fragments into a single chain for more efficient sequencing.

B. To selectively remove unwanted DNA fragments before sequencing for a better distribution.

C. To incorporate chemical modifications that prevent secondary structure formation.

D. To provide binding sites for PCR and enable recognition by the sequencing instrument.

Please note: TopHat questions are ungraded. Engaging honestly with the question will benefit you far more than any shortcuts.

After today, you should have a better understanding of

Principles and innovations of DNA sequencing technologies

Our main problem: Determine the precise ordering of nucleotides

All DNA sequencing technologies are designed to produce a distinct signal corresponding to nucleotides in a specific sequence

Optical: Generated by the interaction of light with nucleotides, often through fluorescence or absorbance.
Electrical: Variations in current or voltage as nucleotides interact with a sensing element.
Chemical: Produced by enzymatic or chemical reactions.

Common signals

After today, you should have a better understanding of

Principles and innovations of DNA sequencing technologies

Chain termination (Sanger)

DNA elongation happens rapidly and continuously

We use DNA polymerase + excess nucleotides to make copies of DNA

Fluorescent tags enable nucleotide detection but require precise signal localization

When excited by light, fluorescent tags emit distinct signals, providing a mechanism to detect nucleotide identity

Issue: How can we determine where the signal is coming from in the sequence?

The length of a DNA fragment can be used to specify a nucleotide location (i.e., the last nucleotide)

3' OH is required for DNA elongation

What happens if we don't have the 3' OH?

We cannot add another nucleotide

Di-deoxynucleotides stop replication

ddNTP will randomly stop DNA elongation

We will be left with DNA strands of variable length with an optical-based signal at the end

When DNA polymerase adds a

ddNTP

, it cannot add any other

nucleotide

Ratio is usually

1

:

100

By sorting DNA fragments by length, we can identify the last nucleotide is

Variable-length fragments

Fragments sorted by length

Last nucleotide order

Original setup

Split DNA sample into four beakers
Add all four dNTPs to each beaker
Add some amount of radioactive ddNTP in a single beaker
Add Taq polymerase and let PCR run

Why would we need separate beakers?

Once we have fragments, how can we separate them by length?

Gel electrophoresis!

Cannot differentiate between radioactive nucleotides

We can build our sequence based on what (radioactive) ddNTP is at that position

Now we use fluorescence to distinguish ddNTPs

Only need one PCR!

We also can automate fragment separation

Capillary gel electrophoresis can accelerate fragment length sorting and detection

Unique fluorescence signal per ddNTP produces a chromatogram

Ideal chromatogram

After today, you should have a better understanding of

Principles and innovations of DNA sequencing technologies

Sequencing by synthesis (Illumina)

Sanger sequencing is highly accurate but lacks scalability and speed for large-scale sequencing

What if we could identify nucleotides as they are being added, allowing us to sequence faster and at a larger scale?

Sequencing by synthesis identifies nucleotides as DNA strands are being synthesized

Immobilizing DNA fragments on a flow cell enables stable signal detection

Bridge amplification generates clusters of identical DNA fragments, amplifying the signal for detection

Bridge amplification creates double-stranded bridges

Clusters will give off a stronger signal compared to a single fragment

Double-stranded clonal bridges are denatured with cleaved reverse strands

Even with immobilization, the signal from a single fragment is often too weak to detect

Forward

Reverse

More on this in later lectures

After today, you should have a better understanding of

Principles and innovations of DNA sequencing technologies

Single molecule sequencing (Nanopore)

Illumina sequencing is cost-effective, scalable, and highly parallel, but limited by short read lengths

Short DNA reads make genome assembly difficult, especially in repetitive regions

Single-molecule sequencing enables long-read sequencing by reading DNA molecules directly

Nanopore sequencing detects nucleotide sequences by measuring changes in ionic current as DNA passes through a pore

DNA passes through a nanopore driven by an electric field.
Each nucleotide disrupts ionic current in a unique, measurable way.
Real-time signal capture translates into nucleotide sequence.

Match each modern sequencing technology with the correct combination of features or characteristics.

Please note: TopHat questions are ungraded. Engaging honestly with the question will benefit you far more than any shortcuts.