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Introduction to library preparation chemistry
Library preparation is the conversion of a DNA or RNA sample to a suitable format for sequencing on Oxford Nanopore Technologies devices. The flow cells used for sequencing the samples contain ion-permeable nanopores embedded in an electrically-resistant membrane enabling an ionic current to pass through the nanopore when a voltage is applied across the membrane. This creates a measurable current that is disrupted when a strand of DNA or RNA passes through the nanopore. The disruption of current is measured and is used to identify the bases passing through the nanopore. Modified bases can also be identified as the nucleic acids are directly sequenced. This means PCR is not required, preventing PCR bias or polymerase error. However, PCR is available to use with our kits to generate more input or repair template damage.
DNA and RNA libraries are prepared by attaching sequencing adapters to strand ends, using either ligation-based or rapid chemistry methods. The sequencing adapters are oligonucleotides that are loaded with a motor protein. The motor protein associates with the nanopore in the flow cell and controls the DNA or RNA strand movement through the nanopores at a defined speed. A hydrophobic tether is also used to localise the template to the membrane into closer contact with the nanopores, improving sensitivity by approximately 10,000 fold.
There are two types of chemistry used in our sequencing kits:
- ligation-based chemistry: The sequencing adapter is ligated onto the DNA ends during library preparation.
- rapid-based chemistry: The sequencing adapter is attached onto the DNA when the transposase cuts the DNA without ligation enzymes. In some kits, adaptation is also be done by PCR.
Find out more information about our sequencing kits in this document.