Biotin labelling covalently attaches biotin to a molecule, making it detectable.

Resulting biotinylated molecule detected via high-affinity binding to streptavidin/avidin.

PCR product contains one biotinylated strand used as template.

Biotin-tagged single-stranded DNA isolated using streptavidin-coated beads.

Template DNA hybridized with a sequencing primer, then added to pyrosequencing reaction.

Reagents (template DNA, enzymes, substrates) loaded.
Addition of nucleotides initiates reaction.
DNA polymerase adds complementary nucleotide, releasing pyrophosphate (PPi).
PPi converted to ATP by ATP sulfurylase (in presence of APS).
The generated ATP converts luciferin to oxyluciferin by luciferase, producing light signals.
Apyrase degrades ATP and unused/unincorporated nucleotides.
Light intensity detected by CCD camera and recorded as peaks.
Reactions:
- DNA template + dNTP (complementary) $\rightarrow$ DNA product + PPi + H$^+$
- PPi + APS $\rightarrow$ ATP + SO$_4^{2-}$
- ATP + luciferin + O$_2$ $\rightarrow$ AMP + PPi + oxyluciferin + CO$_2$ + light
- Unincorporated nucleotide + H$_2$O $\rightarrow$ Nucleoside + Pi

Extract genetic material from samples.
For ultra-long reads, use special methods for high molecular weight DNA (e.g., spin column, magnetic bead, phenol-chloroform).
Fragment extracted DNA (physical shearing, enzymatic digestion).
Optional size selection for specific lengths.
Repair DNA fragment ends for accurate sequencing.
Add adaptors to DNA fragment ends to attach to motor proteins and nanopores.

Library introduced into a flow cell with ionic solution-filled chambers and nanopores.
High concentrations of potassium chloride (KCl) or lithium chloride (LiCl) act as primary ionic solutions.
Constant voltage applied to flow cell creates ionic current through nanopores.
DNA mixed with motor proteins that unwind double helix and transport one strand through nanopore.
Single-stranded DNA passing through nanopore interferes with ionic current.
Each nucleotide causes a specific current change, creating specific signals detected by a patch-clamp amplifier.

Signals translated into DNA sequences using base-calling algorithms.
Base-calling algorithms convert raw signal data into nucleotide sequences (A, C, G, T).
Algorithms interpret signals (electrical current changes, fluorescence colors) to infer base order.
Error correction refines sequence data for accuracy.
Sequenced data aligned to a reference genome, and genome assembly performed.
Structural variants and repetitive regions detected post-assembly and alignment.

Enables analysis of any living thing, by any person, in any environment.
Works in extreme conditions (e.g., -5°C in Antarctica, high humidity in Democratic Republic of Congo).
Used for field studies (e.g., geothermal microbes in Icel