Sample preparation for sequencing........

Library preparation

Clonal amplification

Enrichment ISPs

Sequencing

Data analysis

Library preparation 

Kit contains all reagents 

Fragmentation, end-repair and dA-tailing performed in a single reaction.

Adapter ligation

The DNA fragments are now labeled and any unwanted salts, enzymes and smaller fragments such as leftover adapters are removed.

After pooling the library is ready for clonal amplification. 

Clonal amplification

Each DNA fragment is bound to a single ISP with the P adapter and multiplied by clonal amplification so each ISP is covered with many copies of the same DNA fragment. 

Clonal amplification is performed by an emulsion PCR. The ligated DNA fragments bind to ISPs with the P adapter end. Amplification primers bind to A and P1 adaptors.

At the same time the X adapter is biotinylated to make it possible to enrich for ISPs with DNA. This occurs during the enrichtment step when empty ISPs are removed. At the end you have a sample containing properly amplified ISPs that can be loaded on a chip for sequencing.

Enrichment ISPs

After clonal amplification each Ion Sphere Particle (ISP) contains multiple copies of the same DNA fragment. 

The DNA fragments are bound to the ISP on one side and are biotinylated on the other side. During enrichment the biotinylated side will bind to magnetic beads coated with Streptavidin. So, all empty ISPs that do not contain any DNA fragment are washed away.

The final sample consists of ISPs with DNA fragments and this sample is subsequently loaded onto a chip.

The Ion OneTouch™ ES uses magnetic bead technology to isolate template-positive Ion Sphere™ particles that can be loaded directly onto the Ion semiconductor chip thus delivering automated, highly reproducible enrichment with every run

Beads

All bead-types are applicable to the same type of applications

Choice deopends on the nature of target and sample, the buffers and solutions

To obtain best results, the bead with the size, charge and degree of hydrophobiocity most appropriate for the application should be chosen

Bead surfaces can be hydrophilic or hydrophobic

Based on tosylactivated or carboxylic acid beads

Dynabeads® M-450 Tosylactivated are 4.5 µm superparamagnetic beads containing surface tosyl groups.

Streptavidin is a 52.8 kDa protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin.

Streptavidin Magnetic Beads

Streptavidin Magnetic Beads are 1 µm superparamagnetic particles covalently coupled to a highly pure form of streptavidin.

The beads can be used to capture biotin labeled substrates including antigens, antibodies and nucleic acids 

Streptavidin-coupled Dynabeads™ have been widely used employed in 25,000 routine IVD instruments

Applications include preparing single-stranded DNA templates, isolation of RNA and DNA binding proteins, immobilization of large DNA fragments, purifying sequencing products, and the specific capture of nucleic acids.

Used for sequence specific DNA/RNA capture in nucleic acid diagnostics