Pacific Biosciences vs Oxford Nanopore: The Story of How Scientists Learned to Read Long DNA Molecules
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If you've been following the evolution of DNA sequencing, you've already seen how scientists went from the painstaking days of Sanger sequencing to the high-throughput revolution of next-generation sequencing (NGS). Sequencing that once took days could now be completed in hours, and instead of sequencing a single DNA fragment at a time, millions could be sequenced simultaneously.
It sounded like the perfect solution.
But there was a still room for optimisation. The instruments that enabled millions of sequencing to be performed simultaneously, could only do so with short fragments (150 to 300 base pairs).
While this dramatically increased throughput and reduced cost, it introduced an entirely new challenge. Scientists were no longer reading long stretches of DNA. Instead, they were breaking genomes into millions or even billions of tiny fragments and relying on computers to piece them back together.
Imagine receiving a novel after someone had shredded every page into tiny strips containing only a few words each. You could probably reconstruct many of the sentences. But if the novel contained repeated paragraphs, missing pages, or similar-looking chapters, putting everything back together correctly would become incredibly difficult.
DNA presents exactly the same problem.
Large genomes are full of repetitive DNA sequences. The human genome, for example, is nearly 3.2 billion base pairs long, and almost half of it consists of repetitive elements. If your sequencing reads are only 150 base pairs long, many reads may map equally well to multiple locations within the genome. The computer simply cannot determine where some fragments belong.
This made assembling complete genomes difficult. Structural variants such as large insertions, deletions, inversions, and chromosomal rearrangements were often missed. Highly repetitive regions remained unresolved. Some medically important genes were impossible to assemble accurately.
Scientists needed a way to read much longer pieces of DNA.
Interestingly, two companies approached this challenge in completely different ways.
One company asked:
What if we watched DNA polymerase copy DNA one nucleotide at a time?
The other asked:
What if we didn't copy the DNA at all?
Those two ideas gave rise to two of the most important sequencing technologies available today: Pacific Biosciences' Single Molecule Real-Time (SMRT) sequencing and Oxford Nanopore sequencing.
Although both technologies are classified as long-read sequencing methods, the way they work could hardly be more different.
Pacific Biosciences: Watching DNA Polymerase at Work
Our story begins in the early 2000s.
Scientists had already spent decades studying DNA polymerase, the remarkable enzyme responsible for copying DNA inside every living cell.
Every time a cell divides, DNA polymerase faithfully adds complementary nucleotides to a growing DNA strand.
A pairs with T.
G pairs with C.
Scientists wondered whether this natural process itself could become the sequencing instrument.
Instead of stopping DNA synthesis like Sanger sequencing, or sequencing millions of amplified DNA fragments simultaneously like second-generation sequencing, why not simply watch a single DNA polymerase molecule doing what intelligent design designed it to do?
This became the foundation of Single Molecule Real-Time sequencing, more commonly known as SMRT sequencing.
The name is actually very descriptive.
Single Molecule means that each DNA molecule is sequenced individually without requiring clusters of identical DNA molecules.
Real-Time means the sequencing instrument observes DNA synthesis as it happens.
But this immediately created another problem.
DNA polymerase is an incredibly tiny enzyme. Individual nucleotide incorporation events occur within milliseconds. Even with the world's most sensitive microscopes, how could anyone observe a single enzyme working?
This is where Pacific Biosciences introduced one of the most elegant engineering solutions in modern biotechnology.
Zero-Mode Waveguides: Shrinking the Observation Window
The answer was not to build a better microscope.
Instead, they built a smaller observation chamber.
PacBio developed microscopic structures called Zero-Mode Waveguides, often abbreviated as ZMWs.
A ZMW is an extremely tiny hole etched into a metal film deposited on a glass chip.
These holes are far smaller than the wavelength of visible light.
Normally, light spreads throughout a sample, causing enormous background fluorescence.
Inside a ZMW, however, light cannot propagate normally.
Instead, only an incredibly thin region at the very bottom of the well becomes illuminated.
Think of shining a flashlight into an enormous room versus shining it through the eye of a needle.
Only a tiny volume becomes visible.
This tiny illuminated volume is where the magic happens.
A single DNA polymerase molecule is immobilised at the bottom of each ZMW.
The DNA template is attached to that polymerase.
Everything else remains effectively invisible.
Now scientists could watch one DNA polymerase molecule copying one DNA molecule without being overwhelmed by background fluorescence.
Fluorescent Nucleotides
Next, the polymerase needs nucleotides to build the new DNA strand.
Unlike Sanger sequencing, PacBio does not use chain-terminating nucleotides.
Instead, it uses normal nucleotides carrying fluorescent dyes.
Each of the four nucleotides has its own fluorescent colour.
A emits one colour.
T another.
G another.
C another.
Importantly, the fluorescent dye is attached to the phosphate group rather than the nucleotide base itself.
Why?
Because DNA polymerase naturally removes the phosphate groups during DNA synthesis.
When the nucleotide is incorporated, the fluorescent label is automatically cleaved away and diffuses out of the observation chamber.
The newly synthesised DNA therefore remains completely natural.
This allows the polymerase to continue synthesising DNA uninterrupted for thousands of nucleotides.
Watching DNA Being Copied
Now the sequencing reaction begins.
DNA polymerase selects the next complementary nucleotide.
As that nucleotide briefly sits inside the enzyme before incorporation, its fluorescent dye remains within the illuminated region of the ZMW.
The instrument records a short flash of coloured light.
Once incorporation occurs, the fluorescent label is released.
The light disappears.
Another nucleotide enters.
Another flash occurs.
By recording thousands of these flashes every second, the instrument reconstructs the DNA sequence one nucleotide at a time.
Unlike previous sequencing technologies, PacBio is literally watching DNA synthesis in real time.
But There Was Another Problem
Single-molecule sequencing was ground breaking.
Unfortunately, early versions were not particularly accurate.
Every sequencing technology makes occasional mistakes.
When only one molecule is observed, random errors become much more noticeable.
PacBio needed a way to improve accuracy without sacrificing long read lengths.
The solution was surprisingly clever.
Instead of reading the DNA molecule once...
they decided to read it again.
And again.
And again.
Circular Consensus Sequencing
PacBio converts each DNA fragment into a circle before sequencing.
Special DNA adapters are attached to both ends of the fragment, creating a circular molecule known as a SMRTbell template.
Because the DNA is circular, DNA polymerase simply keeps moving around the same insert multiple times.
Imagine proofreading the same sentence repeatedly.
If you read it once, you might miss a typo.
If you read it ten times, mistakes become much easier to identify.
PacBio applies exactly the same principle.
Each pass around the circular DNA generates another observation of the same sequence.
The instrument combines all these observations into a single consensus sequence.
This approach became known as Circular Consensus Sequencing (CCS).
The resulting reads are called HiFi reads because they combine long read lengths with extremely high accuracy, often exceeding 99.9%.
Today, HiFi sequencing is one of PacBio's defining strengths.
Reading More Than Just DNA Sequence
PacBio's polymerase provides another useful source of information.
DNA polymerase does not move at exactly the same speed across every DNA molecule.
Certain chemical modifications, particularly DNA methylation, cause the polymerase to hesitate slightly before incorporating the next nucleotide.
These subtle changes in incorporation timing can be measured.
Although PacBio is primarily a sequencing platform, it can also detect several DNA modifications by analysing polymerase kinetics.
The Strengths of PacBio
PacBio rapidly became one of the preferred technologies for applications requiring extremely accurate long reads.
Researchers use it for:
High-quality genome assembly
Structural variant detection
Sequencing difficult genomic regions
Full-length transcript sequencing
Haplotype phasing
Clinical research requiring highly accurate long reads
Its biggest strength is simple.
Few sequencing technologies produce reads that are simultaneously long and exceptionally accurate.
Oxford Nanopore Asked a Completely Different Question
While PacBio was perfecting fluorescent sequencing, another group of scientists was thinking very differently.
They asked:
Why copy DNA at all?
After all, DNA already contains the information.
Perhaps the molecule itself could simply be read directly.
This became the foundation of Oxford Nanopore sequencing.
Instead of watching DNA polymerase build a copy, Oxford Nanopore measures the original DNA molecule itself.
What Is a Nanopore?
A nanopore is exactly what its name suggests.
It is an extremely tiny pore only a few nanometres wide.
The pore is formed by a protein embedded within an artificial membrane.
An electrical voltage is applied across the membrane.
This creates a flow of ions through the nanopore, producing a steady electrical current.
Initially, the current remains constant.
Then DNA enters the pore.
Everything changes.
DNA Interrupts the Electrical Current
DNA carries a negative charge because of its phosphate backbone.
When a DNA molecule enters the nanopore, it partially blocks the movement of ions.
The electrical current immediately changes.
Importantly, different combinations of DNA bases obstruct the pore differently.
The nanopore is actually sensing several neighbouring bases simultaneously rather than one nucleotide at a time.
Each group of bases produces its own characteristic electrical signature.
The sequencing instrument continuously records these tiny changes in current thousands of times every second.
Controlling the Speed
At first glance this sounds simple.
But there was another problem.
DNA moves far too quickly.
If allowed to pass freely through the nanopore, the molecule would move so rapidly that meaningful measurements would be impossible.
Oxford Nanopore solved this using another biological enzyme.
A specialised motor protein grabs the DNA and feeds it through the nanopore one small step at a time.
This slows DNA movement sufficiently for the electrical signal to be measured accurately.
Without the motor protein, nanopore sequencing would not work.
Turning Electrical Signals into DNA Sequence
Unlike PacBio, Oxford Nanopore never observes fluorescent light.
Instead, it records one long electrical signal.
The challenge becomes interpreting that signal.
Sophisticated machine learning algorithms analyse the changing current and determine which DNA bases most likely produced each electrical pattern.
This computational process is known as basecalling.
As sequencing chemistry and artificial intelligence have improved, nanopore sequencing accuracy has increased dramatically.
Reading Native DNA and RNA
One of Oxford Nanopore's biggest advantages is that the DNA molecule does not need to be copied.
The original molecule passes directly through the nanopore.
This means researchers can sequence native DNA.
Even more remarkably, Oxford Nanopore can also sequence native RNA directly.
Most sequencing technologies require RNA to be converted into complementary DNA (cDNA) before sequencing.
Nanopore sequencing can often bypass this step entirely.
Detecting DNA Modifications
Because Oxford Nanopore measures the physical properties of the DNA molecule itself, chemical modifications alter the electrical current.
This means methylated DNA produces a different signal from unmethylated DNA.
Researchers can therefore identify many DNA modifications directly during sequencing without requiring additional chemical treatments.
This has made nanopore sequencing particularly valuable for epigenetics research.
Ultra-Long Reads
Perhaps the most famous feature of Oxford Nanopore sequencing is its read length.
In theory, the nanopore does not limit how long a DNA molecule can be.
As long as high-quality DNA can be extracted without breaking, the molecule can continue passing through the pore.
Researchers have generated sequencing reads exceeding one million base pairs in length.
These ultra-long reads are invaluable for assembling highly repetitive genomes that were previously impossible to resolve.
The Strengths of Oxford Nanopore
Oxford Nanopore has carved out a unique position in DNA sequencing.
Its major strengths include:
Ultra-long sequencing reads
Direct sequencing of native DNA
Direct sequencing of native RNA
Detection of DNA modifications
Portable sequencing instruments
Real-time data generation
Perhaps its most recognisable instrument is the MinION, a pocket-sized sequencer small enough to fit in the palm of your hand.
Researchers have used it in remote rainforests, during infectious disease outbreaks, aboard the International Space Station, and in field hospitals where traditional sequencing laboratories simply do not exist.
PacBio vs Oxford Nanopore
Although both technologies generate long reads, they solve the sequencing problem in fundamentally different ways.
PacBio watches DNA polymerase synthesise a new DNA strand.
Oxford Nanopore measures the original DNA molecule passing through a nanopore.
PacBio relies on fluorescent light.
Oxford Nanopore relies on electrical current.
PacBio obtains exceptional accuracy by repeatedly sequencing the same circular DNA molecule to generate HiFi consensus reads.
Oxford Nanopore achieves extraordinary read lengths because the DNA molecule itself passes continuously through the nanopore.
PacBio is generally preferred when the highest possible sequence accuracy is required.
Oxford Nanopore is often preferred when ultra-long reads, portability, direct RNA sequencing, or epigenetic analysis are the primary goals.
Neither technology is universally better.
Each was designed to solve a different problem.
The Future of Long-Read Sequencing
For many years, scientists believed they had to choose between long reads and accurate reads.
Today, that distinction is becoming less clear.
PacBio continues to improve the accuracy and throughput of HiFi sequencing.
Oxford Nanopore continues to improve its nanopore chemistry, pore design, motor proteins, and machine learning basecalling algorithms.
Both technologies are becoming faster, more accurate, and more affordable every year.
Together, they are allowing scientists to study regions of the genome that were previously inaccessible, discover structural variants that short-read sequencing often misses, characterise complex epigenetic modifications, assemble complete genomes with unprecedented accuracy, and even sequence DNA outside traditional laboratories.
The story of long-read sequencing reminds us that there is often more than one solution to the same scientific problem.
Related Reading
New to sequencing technologies? Start with Sanger Sequencing Explained: How One of the Oldest DNA Sequencing Methods Still Works to understand the foundational method that long-read platforms eventually built on.
If you want to see how short-read NGS stacks up against Sanger, check out What Is Next-Generation Sequencing? NGS vs Sanger Explained.
And for a closer look at how Nanopore sequencing compares to the original Sanger method, read What Is Oxford Nanopore Sequencing? How It Differs from Sanger.
